Methods and products for transfecting cells

ABSTRACT

The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods for inducing cells to express proteins and for reprogramming and gene-editing cells using RNA are disclosed. Methods for producing cells from patient samples, cells produced using these methods, and therapeutics comprising cells produced using these methods are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/857,894, filed Apr. 24, 2020, which is a continuation ofU.S. patent application Ser. No. 16/776,765, filed Jan. 30, 2020 (nowU.S. Pat. No. 10,662,410), which is a continuation of U.S. patentapplication Ser. No. 16/567,059, filed Sep. 11, 2019, which is acontinuation of U.S. patent application Ser. No. 16/402,175, filed May2, 2019 (now U.S. Pat. No. 10,472,611), which is a continuation of U.S.patent application Ser. No. 15/429,795, filed Feb. 10, 2017, which is acontinuation of U.S. patent application Ser. No. 15/222,453, filed Jul.28, 2016 (now U.S. Pat. No. 9,605,278), which is a continuation of U.S.patent application Ser. No. 14/296,220, filed Jun. 4, 2014 (now U.S.Pat. No. 9,422,577), which is a continuation of International PatentApplication No. PCT/US2012/067966, filed on Dec. 5, 2012, which claimspriority to U.S. Provisional Application No. 61/566,948, filed on Dec.5, 2011, U.S. Provisional Application No. 61/569,595, filed on Dec. 12,2011, U.S. Provisional Application No. 61/637,570, filed on Apr. 24,2012, and U.S. Provisional Application No. 61/664,494, filed on Jun. 26,2012, which are all hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates in part to nucleic acids encodingproteins, nucleic acids containing non-canonical nucleotides,therapeutics comprising nucleic acids, methods, kits, and devices forinducing cells to express proteins, methods, kits, and devices fortransfecting, gene editing, and reprogramming cells, and cells,organisms, and therapeutics produced using these methods, kits, anddevices.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

This application contains a Sequence Listing in ASCII format submittedelectronically herewith via EFS-Web. Said ASCII copy, created on Jun.17, 2020, is named FAB-001C13_ST25.txt and is 26,783 bytes in size. TheSequence Listing is incorporated herein by reference in its entirety.

BACKGROUND Nucleic-Acid Transfection

Nucleic acids can be delivered to cells both in vitro and in vivo bypre-complexing the nucleic acids with charged lipids, lipidoids,peptides, polymers or mixtures thereof. Such transfection reagents arecommercially available, and are widely used for delivering nucleic acidsto cells in culture. Cells exposed to transfection reagent-nucleic acidcomplexes may internalize these complexes by endocytosis or other means.Once inside a cell, the nucleic acid can carry out its intendedbiological function. In the case of protein-encoding RNA, for example,the RNA can be translated into protein by the ribosomes of the cell.

Serum-Free Cell Culture

Animal sera such as fetal bovine serum (FBS) are commonly used as asupplement in cell-culture media to promote the growth of many types ofcells. However, the undefined nature of serum makes cells that arecontacted with this component undesirable for both research andtherapeutic applications. As a result, serum-free cell-culture mediahave been developed to eliminate the batch-to-batch variability and therisk of contamination with toxic and/or pathogenic substances that areassociated with serum.

The most abundant protein in serum is serum albumin. Serum albumin bindsto a wide variety of molecules both in vitro and in vivo, includinghormones, fatty acids, calcium and metal ions, and small-molecule drugs,and can transport these molecules to cells, both in vitro and in vivo.Serum albumin (most often either bovine serum albumin (BSA) or humanserum albumin (HSA)) is a common ingredient in serum-free cell-culturemedia, where it is typically used at a concentration of 1-10 g/L. Serumalbumin is traditionally prepared from blood plasma by ethanolfractionation (the “Cohn” process). The fraction containing serumalbumin (“Cohn Fraction V” or simply “Fraction V”) is isolated, and istypically used without further treatment. Thus, standard preparations ofserum albumin comprise a protein part (the serum albumin polypeptide)and an associated-molecule part (including salts, fatty acids, etc. thatare bound to the serum albumin polypeptide). The composition of theassociated-molecule component of serum albumin is, in general, complexand unknown.

Serum albumin can be treated for use in certain specialized applications(See Barker A method for the deionization of bovine serum albumin.Tissue Culture Association. 1975; Droge et al. Biochem Pharmacol. 1982;31:3775-9; Ng et al. Nat Protoc. 2008; 3:768-76; U.S. Patent Appl. Pub.No. US 2010/0168000, the contents of which are hereby incorporated byreference). These treatment processes are most commonly used to removeglobulins and contaminating viruses from solutions of serum albumin, andoften include stabilization of the serum albumin polypeptide by additionof the short-chain fatty acid, octanoic acid, followed byheat-inactivation/precipitation of the contaminants. For highlyspecialized stem-cell-culture applications, using an ion-exchange resinto remove excess salt from solutions of BSA has been shown to increasecell viability (See Ng et al. Nat Protoc. 2008; 3:768-76; U.S. PatentAppl. Pub. No. US 2010/0168000, the contents of which are herebyincorporated by reference). However, recombinant serum albumin does notbenefit from such treatment, even in the same sensitivestem-cell-culture applications (See Ng et al. Nat Protoc. 2008;3:768-76; U.S. Patent Appl. Pub. No. US 2010/0168000, the contents ofwhich are hereby incorporated by reference), demonstrating that theeffect of deionization in these applications is to remove excess saltfrom the albumin solution, and not to alter the associated-moleculecomponent of the albumin. In addition, the effect of such treatment onother cell types such as human fibroblasts, and the effect of suchtreatment on transfection efficiency and transfection-associatedtoxicity have not been previously explored. Furthermore,albumin-associated lipids have been shown to be critical for humanpluripotent stem-cell culture, and removing these from albumin has beenshown to result in spontaneous differentiation of human pluripotent stemcells, even when lipids are added separately to the cell-culture medium(See Garcia-Gonzalo et al. PLoS One. 2008; 3:e1384, the contents ofwhich are hereby incorporated by reference). Thus, a cell-culture mediumcontaining albumin with an unmodified associated-molecule component isthought to be critical for the culture of human pluripotent stem cells.Importantly, the relationship between the associated-molecule componentof lipid carriers such as albumin and transfection efficiency andtransfection-associated toxicity has not been previously explored.

Cell Reprogramming

Cells can be reprogrammed by exposing them to specific extracellularcues and/or by ectopic expression of specific proteins, microRNAs, etc.While several reprogramming methods have been previously described, mostthat rely on ectopic expression require the introduction of exogenousDNA, which can carry mutation risks. DNA-free reprogramming methodsbased on direct delivery of reprogramming proteins have been reported,however these methods are too inefficient and unreliable for commercialuse. In addition, RNA-based reprogramming methods have been described,however, existing RNA-based reprogramming methods are slow, unreliable,and inefficient when performed on adult cells, require manytransfections (resulting in significant expense and opportunity forerror), can reprogram only a limited number of cell types, can reprogramcells to only a limited number of cell types, require the use ofimmunosuppressants, and require the use of multiple human-derivedcomponents, including blood-derived HSA and human fibroblast feeders.The many drawbacks of previously disclosed cell-reprogramming methodsmake them undesirable for both research and therapeutic use.

Gene Editing

Several naturally occurring proteins contain DNA-binding domains thatcan recognize specific DNA sequences, for example, zinc fingers (ZFs)and transcription activator-like effectors (TALEs). Fusion proteinscontaining one or more DNA-binding domains and the catalytic domain of anuclease can be used to create a double-strand break in a desired regionof DNA in a cell. When combined with a DNA template containing one ormore regions of homology to the DNA of the cell, gene-editing proteinscan be used to insert a DNA sequence or to otherwise alter the sequenceof the DNA of the cell in a controlled manner. However, most currentmethods for gene editing cells use DNA-based vectors to expressgene-editing proteins. As a result, these gene-editing methods areinefficient, and carry a risk of uncontrolled mutagenesis, making themundesirable for both research and therapeutic use. Methods for DNA-freegene editing of somatic cells have not been previously explored, norhave methods for simultaneous or sequential gene editing andreprogramming of somatic cells. Finally, the use of gene editing in ananti-bacterial, anti-viral, or anti-cancer treatment has not beenpreviously explored.

Model Organisms

Knockout rats have been generated by embryo microinjection of nucleicacids encoding zinc-finger nucleases and TALE-nucleases (TALENs). Geneediting to introduce sequence-specific mutations (a.k.a. “knockins”) hasalso been reported in mice and rats by injecting nucleic acids encodingzinc-finger nucleases into embryos. Genetically-modified rats have beengenerated using embryonic stem cells, and germline-competent ratpluripotent stem cells have been generated by somatic-cellreprogramming. However, the use of gene-edited reprogrammed cells togenerate genetically modified organisms, including mice and rats has notbeen previously explored.

There is a need in the field for improved methods and products fortransfecting cells.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides reagents, protocols, kits,and devices for inducing cells to express proteins and for transfecting,reprogramming, and gene-editing cells. Unlike previously reportedmethods, certain embodiments of the present invention do not involveexposing the cells to exogenous DNA or to allogeneic or animal-derivedmaterials.

In one aspect, the invention provides a synthetic RNA moleculecomprising three or more non-canonical nucleotides that each include oneor more substitutions from the following: pyrimidine position 2C,pyrimidine position 4C, pyrimidine position 5C, purine position 6C,purine position 7N, and purine position 8C. In some embodiments, thesynthetic RNA molecule is produced by in vitro transcription. In otherembodiments, the synthetic RNA molecule further comprises at least oneof: a 5′-cap, a 5′-Cap 1 structure, and a 3′-poly(A) tail. In otherembodiments, at least two of the non-canonical nucleotides arepyrimidines. In still other embodiments, the non-canonical nucleotidesinclude at least one of pseudouridine, 2-thiouridine, 4-thiouridine,5-azauridine, 5-hydroxyuridine, 5-aminouridine, 5-methyluridine,2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine,5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine,5-methylcytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine,5-hydroxycytidine, 5-aminocytidine, N4-methylpseudoisocytidine,2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine,5-aminopseudoisocytidine, 5-methylpseudoisocytidine, N6-methyladenosine,7-deazaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine,6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine,and 6-thio-7-deaza-8-azaguanosine. In other embodiments, at least two ofthe non-canonical nucleotides each comprise less than 20% of thesynthetic RNA molecule. In still other embodiments, the non-canonicalnucleotides include at least one of: pseudouridine, 2-thiouridine,4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-aminouridine,5-methyluridine, 2-thiopseudouridine, 4-thiopseudouridine,5-hydroxypseudouridine, 5-methylpseudouridine, and 5-aminopseudouridine,and at least one of: pseudoisocytidine, 5-methylcytidine,N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine,5-aminocytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine,5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, and5-methylpseudoisocytidine. In a further embodiment, the non-canonicalnucleotides further include at least one of: N6-methyladenosine,7-deazaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine,6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine,and 6-thio-7-deaza-8-azaguanosine.

In another aspect, the invention provides a synthetic RNA molecule thatcomprises a non-canonical nucleotide, and encodes a gene-editingprotein.

In another embodiment, the invention provides a therapeutic compositioncomprising the synthetic RNA molecule described herein.

In another aspect, the invention provides a therapeutic compositioncomprising a synthetic RNA molecule that encodes a gene-editing proteinand a transfection reagent.

In another embodiment, the invention provides a method for transfectinga cell with a nucleic acid comprising contacting the cell with thesynthetic RNA molecule described herein.

In another embodiment, the invention provides a method for inducing amammalian cell to express a protein of interest comprising contactingthe cell with the synthetic RNA molecules described herein.

In another embodiment, the invention provides a method for reprogramminga cell comprising contacting the cell with the synthetic RNA moleculesdescribed herein. In another embodiment, the invention provides a methodfor gene-editing a cell comprising contacting the cell with thesynthetic RNA molecules described herein.

In another aspect, the invention provides a method for transfecting acell with a nucleic acid comprising: contacting the cell with a mediumcontaining hydrocortisone and/or albumin, wherein the albumin is treatedwith an ion-exchange resin or charcoal, and contacting the cell with thenucleic acid. In one embodiment, the albumin is treated with ashort-chain fatty acid, and/or brought to a temperature of at least 40°C. In other embodiments, the method further comprises contacting thecell with a transfection reagent. In other embodiments, the cell is amammalian cell, and the mammalian cell is induced to express a proteinof interest. In other embodiments, the method further comprisescontacting the cell with the nucleic acid at least twice during 5consecutive days. In some embodiments, the nucleic acid encodes areprogramming protein. In other embodiments, the cell is reprogrammed.In yet another embodiment, the cell is a skin cell, and furthercomprising culturing the skin cell under conditions that support thegrowth of at least one of: skin cells, pluripotent stem cells,glucose-responsive insulin-producing cells, hematopoietic cells, cardiaccells, and retinal cells, and wherein the skin cell is reprogrammed to acell selected from: a skin cell, a pluripotent stem cell, aglucose-responsive insulin-producing cell, a hematopoietic cell, acardiac cell, and a retinal cell. In yet another embodiment, the nucleicacid encodes Oct4 protein. In yet another embodiment, the method furthercomprises contacting the cell with a nucleic acid that encodes at leastone of: Sox2 protein, Klf4 protein, and c-Myc protein. In yet anotherembodiment, the method further comprises contacting the cell with one ormore nucleic acids that encode Sox2 protein, Klf4 protein, and c-Mycprotein. In still other embodiments, the nucleic acid encodes agene-editing protein. In still other embodiments, the nucleic acidencodes a protein that, acting alone or in combination with one or moreother molecules, creates a single-strand or double-strand break in a DNAmolecule. In various embodiments, the cell is gene-edited. In someembodiments, the single-strand or double-strand break is within about5,000,000 bases of the transcription start site of a gene selected from:CCR5, CXCR4, GAD1, GAD2, CFTR, HBA1, HBA2, HBB, HBD, FANCA, XPA, XPB,XPC, ERCC2, POLH, HTT, DMD, SOD1, APOE, APP, LRRK2, PRNP, BRCA1, andBRCA2 or an analogue, variant or family-member thereof. In someembodiments, the method further comprises contacting the cell with atleast one of: poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin,vitronectin, collagen, and laminin, or a biologically active fragment,functional variant or family-member thereof. In still other embodiments,the nucleic acid is a synthetic RNA molecule, which may contain at leastone of: pseudouridine, 5-methylpseudouridine, and 5-methylcytidine. Insome embodiments, the method provides for contacting the cell with adifferentiation factor and/or harvesting the cell from a patient and/ordelivering the cell to a patient.

In another aspect, the invention provides a medium comprising albumin,wherein the albumin is recombinant, and treated with an ion-exchangeresin or charcoal. In another embodiment, the medium further comprises abuffered salt solution and amino acids and/or one or more of insulin,transferrin, and selenium and/or cholesterol and/or a steroid (such as,for example, hydrocortisone) and/or an immunosuppressant (such as, forexample, B18R).

In another aspect, the invention provides a kit comprisinghydrocortisone and/or albumin, wherein the albumin is treated with anion-exchange resin or charcoal, and a synthetic RNA molecule. In oneembodiment, the synthetic RNA molecule encodes at least one of: Oct4protein, Sox2 protein, Klf4 protein, c-Myc protein, Nanog protein, Lin28protein, and Utf1 protein. In another embodiment, the kit furthercomprises a transfection reagent and/or the synthetic RNA moleculesdescribed herein. In another embodiment, the kit is a reprogramming kitand/or a gene-editing kit.

In another aspect, the invention provides a nucleic acidtransfection-reagent complex comprising a nucleic acid and atransfection reagent, wherein the nucleic acid transfection-reagentcomplex is solidified by cooling. In some embodiments, the nucleic acidtransfection-reagent complex is solidified by contacting the nucleicacid transfection-reagent complex with liquid nitrogen in the liquidand/or vapor phase.

In another aspect, the invention provides a method for transfecting acell comprising contacting the cell with the nucleic acidtransfection-reagent complex described herein.

In another aspect, the invention provides a system for transfectingcells comprising a means for contacting cells with a transfection mediumand a means for contacting the cells with nucleic acidtransfection-reagent complexes. In some embodiments, the atmospherearound the cells contains approximately 5% carbon dioxide and/orapproximately 5% oxygen.

In some embodiments, the invention provides a cell and/or an organismand/or a therapeutic composition and/or a therapeutic compositioncomprising a cell produced by the methods described herein.

In some aspects, synthetic RNA molecules with low toxicity and hightranslation efficiency are provided. In other aspects, methods, kits,and devices for producing and delivering synthetic RNA molecules tocells are provided. In still other aspects, a cell-culture medium forhigh-efficiency transfection, reprogramming, and gene editing of cellsis provided. Other aspects relate to therapeutics comprising syntheticRNA molecules, including for the treatment of type 1 diabetes, heartdisease, including ischemic and dilated cardiomyopathy, maculardegeneration, Parkinson's disease, cystic fibrosis, sickle-cell anemia,thalassemia, Fanconi anemia, severe combined immunodeficiency,hereditary sensory neuropathy, xeroderma pigmentosum, Huntington'sdisease, muscular dystrophy, amyotrophic lateral sclerosis, Alzheimer'sdisease, cancer, and infectious diseases including hepatitis andHIV/AIDS. Further aspects relate to therapeutics comprising cells,including for the treatment of type 1 diabetes, heart disease, includingischemic and dilated cardiomyopathy, macular degeneration, Parkinson'sdisease, cystic fibrosis, sickle-cell anemia, thalassemia, Fanconianemia, severe combined immunodeficiency, hereditary sensory neuropathy,xeroderma pigmentosum, Huntington's disease, muscular dystrophy,amyotrophic lateral sclerosis, Alzheimer's disease, cancer, andinfectious diseases including hepatitis and HIV/AIDS.

DETAILED DESCRIPTION OF THE FIGURES

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings and in which:

FIG. 1 depicts RNA encoding the indicated proteins, resolved on adenaturing formaldehyde-agarose gel.

FIG. 2A depicts primary human fibroblasts, transfected with syntheticRNA encoding Oct4 and comprising the indicated nucleotides. “A” refersto adenosine, “G” refers to guanosine, “U” refers to uridine, “C” refersto cytidine, “psU” refers to pseudouridine, “5mC” refers to5-methylcytidine, “N4mC” refers to N4-methylcytidine, “7dG” refers to7-deazaguanosine, and “psisoC” refers to pseudoisocytidine. Numberspreceding nucleotides indicate the fraction of the correspondingnucleotide-5′-triphosphate in the in vitro-transcription reaction. Forexample, 0.5 N4mC refers to RNA synthesized in an in vitro-transcriptionreaction containing equal amounts of N4-methylcytidine-5′-triphosphateand cytidine-5′-triphosphate. Cells were fixed and stained for Oct4protein 20 h after transfection.

FIG. 2B depicts Oct4 expression and cell density of cultures of primaryhuman fibroblasts, transfected with synthetic RNA encoding Oct4 andcomprising the indicated nucleotides. Nucleotides are abbreviated as inFIG. 2A, except that “7dA” refers to 7-deazaadenosine, and “piC” refersto pseudoisocytidine. Cell density is shown normalized to untransfectedcells. Oct4 expression is shown normalized to synthetic RNA containingonly canonical nucleotides. Error bars indicate the standard error(n=3).

FIG. 3A depicts a reprogrammed cell line, generated by transfectingprimary human fibroblasts with RNA encoding the proteins Oct4, Sox2,Klf4, c-Myc-2 (T58A), and Lin28, one day after colonies were picked andplated on a basement membrane extract-coated plate.

FIG. 3B depicts a reprogrammed cell line, generated as in FIG. 3A,stained for the pluripotent stem-cell markers Oct4 and SSEA4. The panellabeled “Hoechst” shows the nuclei, and the panel labeled “Merge” showsthe merged signals from the three channels.

FIG. 3C depicts primary human fibroblasts, transfected and cultured asin FIG. 3A. A total of 5 transfections were performed. Pictures weretaken on day 7. Several colonies of cells with a reprogrammed morphologyare visible.

FIG. 4A depicts a 1.5 mm-diameter dermal punch biopsy tissue sample.

FIG. 4B depicts a tissue sample, harvested as in FIG. 4A, and suspendedat the air-liquid interface of a solution containing an enzyme.

FIG. 4C depicts primary human fibroblasts, harvested as in FIG. 4A,dissociated as in FIG. 4B, and plated in a well of a 96-well plate.

FIG. 5A depicts primary human fibroblasts, reprogrammed toinsulin-producing cells. Cells were fixed and stained for insulin.

FIG. 5B depicts primary human fibroblasts, reprogrammed to hematopoieticcells. Cells were fixed and stained for CD34.

FIG. 5C depicts primary human fibroblasts, reprogrammed to beatingcardiac cells.

FIG. 6A depicts the forward strand of an in vitro-transcription templatefor producing an RNA TALEN™ backbone.

FIG. 6B depicts the template of FIG. 6A after a Golden-Gate cloningreaction to incorporate a series of monomer repeats, forming a completeRNA TALEN™ template.

FIG. 6C depicts a 5′-capped, 3′-poly(A)-tailed RNA TALEN™ produced fromthe template of FIG. 6B.

FIG. 7 depicts the sequence of the template of FIG. 6B, wherein the RNATALEN™ is designed to bind to a 20 bp region of DNA, and wherein theregions labeled “X(02)X”, “X(03)X”, and so forth, represent the repeatvariable domains (RVDs) that can be selected to target a specific DNAsequence. This template encodes an RNA TALEN™, wherein the first residuebound by the RNA TALEN™ is a thymidine residue, irrespective of theRVDs, and thus the first RVD is labeled “X(02)X” instead of “X(01)X”.

FIG. 8 depicts primary human fibroblasts, gene-edited and reprogrammed.Arrows indicate colonies of cells with a reprogrammed morphology.

FIG. 9A depicts the front view of a system that can transfect and/orreprogram cells in an automated or semi-automated manner.

FIG. 9B depicts the back panel of the system of FIG. 9A.

FIG. 9C depicts major components of the system of FIG. 9A.

FIG. 10A depicts the complexation of RNA and a transfection reagentwithin a complexation medium.

FIG. 10B depicts two methods for dispensing pre-complexed pelletscontaining nucleic acids.

FIG. 10C depicts a method for removing the lid from a well plate usingsuction.

FIG. 10D depicts a method for removing the lid from a well plate using agripper.

FIG. 11 depicts a system that can transfect and/or reprogram cells in anautomated or semi-automated manner in operable combination withequipment for imaging, incubating, and otherwise manipulating the cells.

DEFINITIONS

By “molecule” is meant a molecular entity (molecule, ion, complex,etc.).

By “protein” is meant a polypeptide.

By “RNA molecule” is meant a molecule that comprises RNA.

By “synthetic RNA molecule” is meant an RNA molecule that is producedoutside of a cell or that is produced inside of a cell usingbioengineering, for example, an RNA molecule that is produced in an invitro-transcription reaction, an RNA molecule that is produced by directchemical synthesis or an RNA molecule that is produced in agenetically-engineered E. coli cell.

By “nucleotide” is meant a nucleotide or a fragment or derivativethereof, for example, a nucleobase, a nucleoside, anucleotide-triphosphate, etc.

By “nucleoside” is meant a nucleotide.

By “transfection” is meant contacting a cell with a molecule, whereinthe molecule is internalized by the cell.

By “upon transfection” is meant during or after transfection.

By “transfection reagent” is meant a substance or mixture of substancesthat associates with a molecule and facilitates the delivery of themolecule to and/or internalization of the molecule by a cell, forexample, a cationic lipid, a charged polymer or a cell-penetratingpeptide.

By “reagent-based transfection” is meant transfection using atransfection reagent.

By “cell-culture medium” is meant a medium that can be used for cellculture, for example, Dulbecco's Modified Eagle's Medium (DMEM) orDMEM+10% fetal bovine serum (FBS).

By “complexation medium” is meant a medium to which a transfectionreagent and a molecule to be transfected are added and in which thetransfection reagent associates with the molecule to be transfected.

By “transfection medium” is meant a medium that can be used fortransfection, for example, Dulbecco's Modified Eagle's Medium (DMEM) orDMEM/F12.

By “recombinant protein” is meant a protein or peptide that is notproduced in animals or humans. Non-limiting examples include humantransferrin that is produced in bacteria, human fibronectin that isproduced in an in vitro culture of mouse cells, and human serum albuminthat is produced in a rice plant.

By “lipid carrier” is meant a substance that can increase the solubilityof a lipid or lipid-soluble molecule in an aqueous solution, forexample, human serum albumin or methyl-beta-cyclodextrin.

By “Oct4 protein” is meant a protein that is encoded by the POU5F1 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, for example, human Oct4 protein (SEQ IDNO:1), mouse Oct4 protein, Oct1 protein, a protein encoded by POU5F1pseudogene 2, a DNA-binding domain of Oct4 protein or an Oct4-GFP fusionprotein. In some embodiments the Oct4 protein comprises an amino acidsequence that has at least 70% identity with SEQ ID NO:1, or in otherembodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ IDNO:1. In some embodiments, the Oct4 protein comprises an amino acidsequence having from 1 to 20 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO:1. In otherembodiments, the Oct4 protein comprises an amino acid sequence havingfrom 1 to 15 or from 1 to 10 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO:1.

By “Sox2 protein” is meant a protein that is encoded by the SOX2 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, for example, human Sox2 protein (SEQ IDNO:2), mouse Sox2 protein, a DNA-binding domain of Sox2 protein or aSox2-GFP fusion protein. In some embodiments the Sox2 protein comprisesan amino acid sequence that has at least 70% identity with SEQ ID NO:2,or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identitywith SEQ ID NO:2. In some embodiments, the Sox2 protein comprises anamino acid sequence having from 1 to 20 amino acid insertions,deletions, or substitutions (collectively) with respect to SEQ ID NO:2.In other embodiments, the Sox2 protein comprises an amino acid sequencehaving from 1 to 15 or from 1 to 10 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO:2.

By “Klf4 protein” is meant a protein that is encoded by the KLF4 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, for example, human Klf4 protein (SEQ IDNO:3), mouse Klf4 protein, a DNA-binding domain of Klf4 protein or aKlf4-GFP fusion protein. In some embodiments the Klf4 protein comprisesan amino acid sequence that has at least 70% identity with SEQ ID NO:3,or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identitywith SEQ ID NO:3. In some embodiments, the Klf4 protein comprises anamino acid sequence having from 1 to 20 amino acid insertions,deletions, or substitutions (collectively) with respect to SEQ ID NO:3.In other embodiments, the Klf4 protein comprises an amino acid sequencehaving from 1 to 15 or from 1 to 10 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO:3.

By “c-Myc protein” is meant a protein that is encoded by the MYC gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, for example, human c-Myc protein (SEQ IDNO:4), mouse c-Myc protein, 1-Myc protein, c-Myc (T58A) protein, aDNA-binding domain of c-Myc protein or a c-Myc-GFP fusion protein. Insome embodiments the c-Myc protein comprises an amino acid sequence thathas at least 70% identity with SEQ ID NO:4, or in other embodiments, atleast 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO:4. In someembodiments, the c-Myc protein comprises an amino acid having from 1 to20 amino acid insertions, deletions, or substitutions (collectively)with respect to SEQ ID NO:4. In other embodiments, the c-Myc proteincomprises an amino acid sequence having from 1 to 15 or from 1 to 10amino acid insertions, deletions, or substitutions (collectively) withrespect to SEQ ID NO:4.

By “reprogramming” is meant causing a change in the phenotype of a cell,for example, causing a β-cell progenitor to differentiate into a matureβ-cell, causing a fibroblast to dedifferentiate into a pluripotent stemcell, causing a keratinocyte to transdifferentiate into a cardiac stemcell or causing the axon of a neuron to grow.

By “reprogramming factor” is meant a molecule that, when a cell iscontacted with the molecule and/or the cell expresses the molecule, can,either alone or in combination with other molecules, causereprogramming, for example, Oct4 protein.

By “feeder” is meant a cell that can be used to condition medium or tootherwise support the growth of other cells in culture.

By “conditioning” is meant contacting one or more feeders with a medium.

By “fatty acid” is meant a molecule that comprises an aliphatic chain ofat least two carbon atoms, for example, linoleic acid, α-linolenic acid,octanoic acid, a leukotriene, a prostaglandin, cholesterol, aglucocorticoid, a resolvin, a protectin, a thromboxane, a lipoxin, amaresin, a sphingolipid, tryptophan, N-acetyl tryptophan or a salt,methyl ester or derivative thereof.

By “short-chain fatty acid” is meant a fatty acid that comprises analiphatic chain of between two and 30 carbon atoms.

By “albumin” is meant a protein that is highly soluble in water, forexample, human serum albumin.

By “associated molecule” is meant a molecule that is non-covalentlybound to another molecule.

By “associated-molecule-component of albumin” is meant one or moremolecules that are bound to an albumin polypeptide, for example, lipids,hormones, cholesterol, calcium ions, etc. that are bound to an albuminpolypeptide.

By “treated albumin” is meant albumin that is treated to reduce, remove,replace or otherwise inactivate the associated-molecule-component of thealbumin, for example, human serum albumin that is incubated at anelevated temperature, human serum albumin that is contacted with sodiumoctanoate or human serum albumin that is contacted with a porousmaterial.

By “ion-exchange resin” is meant a material that, when contacted with asolution containing ions, can replace one or more of the ions with oneor more different ions, for example, a material that can replace one ormore calcium ions with one or more sodium ions.

By “germ cell” is meant a sperm cell or an egg cell.

By “pluripotent stem cell” is meant a cell that can differentiate intocells of all three germ layers (endoderm, mesoderm, and ectoderm) invivo.

By “somatic cell” is meant a cell that is not a pluripotent stem cell ora germ cell, for example, a skin cell.

By “glucose-responsive insulin-producing cell” is meant a cell that,when exposed to a certain concentration of glucose, can produce and/orsecrete an amount of insulin that is different from (either less than ormore than) the amount of insulin that the cell produces and/or secreteswhen the cell is exposed to a different concentration of glucose, forexample, a β-cell.

By “hematopoietic cell” is meant a blood cell or a cell that candifferentiate into a blood cell, for example, a hematopoietic stem cellor a white blood cell.

By “cardiac cell” is meant a heart cell or a cell that can differentiateinto a heart cell, for example, a cardiac stem cell or a cardiomyocyte.

By “retinal cell” is meant a cell of the retina or a cell that candifferentiate into a cell of the retina, for example, a retinalpigmented epithelial cell.

By “skin cell” is meant a cell that is normally found in the skin, forexample, a fibroblast, a keratinocyte, a melanocyte, an adipocyte, amesenchymal stem cell, an adipose stem cell or a blood cell.

By “Wnt signaling agonist” is meant a molecule that can perform one ormore of the biological functions of one or more members of the Wntfamily of proteins, for example, Wnt1, Wnt2, Wnt3, Wnt3a or2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine.

By “IL-6 signaling agonist” is meant a molecule that can perform one ormore of the biological functions of IL-6 protein, for example, IL-6protein or IL-6 receptor (also known as soluble IL-6 receptor, IL-6R,IL-6R alpha, etc.).

By “TGF-β signaling agonist” is meant a molecule that can perform one ormore of the biological functions of one or more members of the TGF-βsuperfamily of proteins, for example, TGF-β1, TGF-β3, Activin A, BMP-4or Nodal.

By “immunosuppressant” is meant a substance that can suppress one ormore aspects of an immune system, and that is not normally present in amammal, for example, B18R or dexamethasone.

By “gene editing” is meant altering the DNA sequence of a cell.

By “gene-editing protein” is meant a protein that can, either alone orin combination with another molecule, alter the DNA sequence of a cell,for example, a nuclease, a transcription activator-like effectornuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase or anatural or engineered variant, family-member, orthologue, fragment orfusion construct thereof

By “single-strand break” is meant a region of single-stranded ordouble-stranded DNA in which one or more of the covalent bonds linkingthe nucleotides has been broken in one of the one or two strands.

By “double-strand break” is meant a region of double-stranded DNA inwhich one or more of the covalent bonds linking the nucleotides has beenbroken in each of the two strands.

Serum albumin is a common component of serum-free cell-culture media. Ithas now been discovered that serum albumin can inhibit transfection, andthat including untreated serum albumin in a transfection medium atconcentrations normally used in serum-free cell-culture media can resultin low transfection efficiency and/or low cell viability upontransfection. The serum albumin polypeptide can bind to a wide varietyof molecules, including lipids, ions, cholesterol, etc., both in vitroand in vivo, and as a result, both serum albumin that is isolated fromblood and recombinant serum albumin comprise a polypeptide component andan associated-molecule component. It has now been discovered that thelow transfection efficiency and low cell viability upon transfectioncaused by serum albumin can be caused in part by the associated-moleculecomponent of the serum albumin. It has been further discovered thattransfection efficiency can be increased and transfection-associatedtoxicity can be reduced by partially or completely reducing, removing,replacing or otherwise inactivating the associated-molecule component ofserum albumin. Certain embodiments of the invention are thereforedirected to a method for treating a protein to partially or completelyreduce, remove, replace or otherwise inactivate the associated-moleculecomponent of the protein. Other embodiments are directed to a proteinthat is treated to partially or completely reduce, remove, replace orotherwise inactivate the associated-molecule component of the protein.

Certain embodiments are directed to a method for treating a protein bycontacting the protein with one or more molecules that reduce the lowtransfection efficiency and/or low cell viability upon transfectioncaused by the protein. Contacting serum albumin with the short-chainfatty acid, sodium octanoate (also known as “octanoic acid”,“octanoate”, “caprylate” or “caprylic acid”) was found to reduce the lowtransfection efficiency and low cell viability upon transfection causedby serum albumin in certain situations. Other substances that can beused to treat a protein include: capric acid, lauric acid, myristicacid, palmitic acid, stearic acid, arachidic acid, behenic acid,lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid,sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid,linoelaidic acid, alpha-linolenic acid, arachidonic acid,eicosapentaenoic acid, erucic acid, docosahexaenoic acid, tryptophan,N-acetyl tryptophan, cholesterol, other fatty acids, and salts,mixtures, fragments, and derivatives thereof. Substances for treating aprotein can be pure substances, well-defined mixtures or complex orundefined mixtures such as animal-based or plant-based oils, forexample, cod-liver oil. In certain embodiments, a protein is treatedafter the protein is purified. In other embodiments, a protein istreated before the protein is purified. In still other embodiments, aprotein is treated at the same time that the protein is purified. Instill other embodiments, a protein is treated, and the protein is notpurified.

Incubating a protein at an elevated temperature can cause partial orcomplete denaturation of the polypeptide component of the protein, whichcan reduce or eliminate binding sites that may be critical tomaintaining the associated-molecule component of the protein. Certainembodiments are therefore directed to a method for treating a protein byincubating the protein at an elevated temperature. In one embodiment,the protein is incubated at a temperature of at least about 40° C. forat least about 10 minutes. In another embodiment, the protein isincubated at a temperature of at least about 50° C. for at least about10 minutes. In another embodiment, the protein is incubated at atemperature of at least about 55° C. for at least about 30 minutes. Inone embodiment, the protein is contacted with sodium octanoate, and thenincubated at about 60° C. for several hours, such as between about 1hour and about 24 hours, or between about 2 hours and about 6 hours. Inanother embodiment, the concentration of sodium octanoate is betweenabout 5 mM and about 50 mM, or between about 10 mM and about 40 mM. Incertain embodiments, the sodium octanoate is replaced with or used incombination with at least one element of capric acid, lauric acid,myristic acid, palmitic acid, stearic acid, arachidic acid, behenicacid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid,sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid,linoelaidic acid, alpha-linolenic acid, arachidonic acid,eicosapentaenoic acid, erucic acid, docosahexaenoic acid, tryptophan,N-acetyl tryptophan, and cholesterol or a salt, mixture, fragment, andderivative thereof.

Glycation and glycosylation are processes by which one or more sugarmolecules are bound to a protein. Glycation and glycosylation can impactthe binding properties of a protein, and serum albumin contains severalpotential glycation sites. Certain embodiments are therefore directed toa method for treating a protein by glycating or glycosylating theprotein.

Ion-exchange resins, including anion-exchange, cation-exchange, andmixed-bed resins, are routinely used to deionize solutions. Theassociated-molecule component of proteins such as serum albumin cancomprise ions. Certain embodiments are therefore directed to a methodfor treating a protein by contacting the protein with one or moreion-exchange resins. In one embodiment, the one or more ion-exchangeresins includes a mixed-bed resin containing functional groups withproton (H+) and hydroxyl (OH−) forms. In another embodiment, the one ormore ion-exchange resins includes an indicator that changes color as theresin becomes saturated with ions. In addition to contacting with one ormore ion-exchange resins, other methods can be used to reduce, remove,replace or otherwise inactivate the associated-molecule component of aprotein, including contacting the protein with charcoal, which may beactivated and/or treated with a chemical such as dextran sulfate,dialysis (including dilution resulting in de-association of theassociated-molecule component, whether or not the de-associatedmolecules are subsequently removed from the solution), crystallization,chromatography, electrophoresis, heat treatment, low-temperaturetreatment, high-pH treatment, low-pH treatment, organic-solventprecipitation, and affinity purification.

Certain methods for treating a protein may preferentially reduce,remove, replace or otherwise inactivate specific types of molecules. Incertain situations, it can therefore be beneficial to combine two ormore methods for treating a protein to reduce the low transfectionefficiency and/or low cell viability upon transfection caused by theprotein. Certain embodiments are therefore directed to a method fortreating a protein using two or more methods to reduce, remove, replaceor otherwise inactivate the associated-molecule component of theprotein. In one embodiment, a protein is contacted with one or moreion-exchange resins and activated charcoal. In another embodiment, aprotein is contacted with sodium octanoate, incubated at an elevatedtemperature, contacted with one or more ion-exchange resins, andcontacted with activated charcoal. In another embodiment, the protein isserum albumin, and the elevated temperature is at least about 50° C.

Certain elements of the associated-molecule component of a protein canbe beneficial to cells in culture, and/or to transfection, for example,certain resolvins, protectins, lipoxins, maresins, eicosanoids,prostacyclins, thromboxanes, leukotrienes, cyclopentenoneprostaglandins, and glucocorticoids. Certain embodiments are thereforedirected to a method for treating a protein to reduce, remove, replaceor otherwise inactivate the associated-molecule component of the proteinwithout reducing, removing, replacing or otherwise inactivating one ormore beneficial elements of the associated-molecule component of theprotein. Other embodiments are directed to a method for treating aprotein to reduce, remove, replace or otherwise inactivate theassociated-molecule component of the protein, and further contacting theprotein with one or more molecules comprising one or more beneficialelements of the associated-molecule component of the protein.

Still other embodiments are directed to a method for treating a proteinto reduce the low transfection efficiency and/or low cell viability upontransfection caused by the protein by contacting the protein with one ormore molecules comprising one or more beneficial elements of theassociated-molecule component of the protein. Still other embodimentsare directed to a method for increasing transfection efficiency and/orincreasing cell viability upon transfection by contacting a cell withone or more molecules comprising one or more beneficial elements of theassociated-molecule component of a protein. In one embodiment, theprotein is contacted with one or more ion-exchange resins or charcoal,and is further contacted with a glucocorticoid, such as hydrocortisone,prednisone, prednisolone, methylprednisolone, dexamethasone orbetamethasone. In another embodiment, the cell is contacted with aglucocorticoid, such as hydrocortisone, prednisone, prednisolone,methylprednisolone, dexamethasone or betamethasone. It has been furtherdiscovered that in certain situations, including one or more steroidsand/or one or more antioxidants in the transfection medium can increasetransfection efficiency, reprogramming efficiency, and gene-editingefficiency. Certain embodiments are therefore directed to a method forinducing a cell to express a protein of interest by culturing the cellin a medium containing a steroid and contacting the cell with one ormore synthetic RNA molecules. In one embodiment, the steroid ishydrocortisone. In another embodiment, the hydrocortisone is present inthe medium at a concentration of between about 0.1 uM and about 10 uM,or about 1 uM. Other embodiments are directed to a method for inducing acell to express a protein of interest by culturing the cell in a mediumcontaining an antioxidant and contacting the cell with one or moresynthetic RNA molecules. In one embodiment, the antioxidant is ascorbicacid or ascorbic-acid-2-phosphate. In another embodiment, the ascorbicacid or ascorbic-acid-2-phosphate is present in the medium at aconcentration of between about 0.5 mg/L and about 500 mg/L, includingabout 50 mg/L. Still other embodiments are directed to a method forreprogramming and/or gene-editing a cell by culturing the cell in amedium containing a steroid and/or an antioxidant and contacting thecell with one or more synthetic RNA molecules, wherein the one or moresynthetic RNA molecules encodes one or more reprogramming and/orgene-editing proteins. In certain embodiments, the cell is present in anorganism, and the steroid and/or antioxidant are delivered to theorganism.

Adding transferrin to the complexation medium has been reported toincrease the efficiency of plasmid transfection in certain situations.It has now been discovered that adding transferrin to the complexationmedium can also increase the efficiency of transfection with syntheticRNA molecules. Certain embodiments are therefore directed to a methodfor inducing a cell to express a protein of interest by adding one ormore synthetic RNA molecules and a transfection reagent to a solutioncontaining transferrin. In one embodiment, the transferrin is present inthe solution at a concentration of between about 1 mg/L and about 100mg/L, such as about 5 mg/L. In another embodiment, the transferrin isrecombinant.

Other embodiments are directed to a medium containing a protein that istreated according to one or more of the methods of the presentinvention. In certain embodiments, the protein is treated before beingmixed with one or more of the other ingredients of the medium. In oneembodiment, the medium is a transfection medium. In another embodiment,the medium also supports efficient transfection and high cell viability.In certain embodiments, the protein and one or more molecules thatreduce the low transfection efficiency and/or low cell viability upontransfection caused by the protein are added independently to themedium. In one embodiment, the protein is treated before being mixedwith one or more of the other ingredients of the medium. In anotherembodiment, the medium is prepared by first treating a concentratedsolution of serum albumin by contacting the concentrated solution ofserum albumin with one or more ion-exchange resins, then removing theone or more ion-exchange resins from the concentrated solution of serumalbumin, and then adding the treated concentrated solution of serumalbumin to the other components of the medium. In another embodiment,the concentrated solution of serum albumin is further contacted withcharcoal before adding the concentrated solution of serum albumin to theother components of the medium. In still another embodiment, theconcentrated solution of serum albumin is first contacted with sodiumoctanoate, then raised to a temperature of at least about 50° C. for atleast about 10 minutes, then contacted with one or more ion-exchangeresins, then contacted with activated charcoal, and then added to theother components of the medium.

It has now been discovered that transfecting cells using a mediumcontaining a buffered salt solution, amino acids, cholesterol,hydrocortisone, and serum albumin can result in efficient transfection,and that transfecting cells using a medium consisting essentially of abuffered salt solution, amino acids, insulin, transferrin, cholesterol,hydrocortisone, serum albumin, and a fibroblast growth factor can resultin efficient transfection and efficient reprogramming. Certainembodiments are therefore directed to a transfection medium containing:a buffered salt solution, amino acids, cholesterol, hydrocortisone, andserum albumin. Other embodiments are directed to a transfection mediumconsisting essentially of and/or comprising: a buffered salt solution,amino acids, insulin, transferrin, cholesterol, hydrocortisone, serumalbumin, and a fibroblast growth factor. Still other embodiments aredirected to a reprogramming medium consisting essentially of and/orcomprising: a buffered salt solution, amino acids, insulin, transferrin,cholesterol, hydrocortisone, serum albumin, and a fibroblast growthfactor. In one embodiment, the medium also includespolyoxyethylenesorbitan monooleate and/or D-alpha-tocopherol acetate. Inanother embodiment, the medium also includes ascorbic acid orascorbic-acid-2-phosphate, for example, at a concentration of betweenabout 1 mg/L and about 100 mg/L. In one embodiment, the hydrocortisoneis present at a concentration of about 1 uM. In another embodiment, thefibroblast growth factor is basic fibroblast growth factor, and thebasic fibroblast growth factor is present at a concentration of betweenabout 1 ng/mL and about 200 ng/mL, such as between about 4 ng/mL andabout 100 ng/mL, or between about 10 ng/mL and about 50 ng/mL, or about20 ng/mL. In one embodiment, the serum albumin is human serum albumin,and the human serum albumin is present at a concentration of betweenabout 0.05% and about 2%, including between about 0.1% and about 1%,such as about 0.5%. In another embodiment, the human serum albumin isrecombinant. In yet another embodiment, the cholesterol is present at aconcentration of about 4.5 mg/L. In one embodiment, the medium does notcontain any animal-derived components. In another embodiment, the mediumdoes not contain any undefined components, for example, cod liver-oilfatty acids or serum. In one embodiment, the medium contains a TGF-βinhibitor, for example, A83-01 or SB431542. In one embodiment, the TGF-βinhibitor is present at a concentration of between about 0.1 uM andabout 10 uM. In one embodiment, the medium contains a Wnt signalingagonist, such as Wnt3a. In another embodiment, the Wnt signaling agonistis present at a concentration of between about 10 ng/mL and about 500ng/mL, including between about 50 ng/mL and about 200 ng/mL. In oneembodiment, the medium contains a source of selenium, such as sodiumselenite.

In certain situations, it may be desirable to replace animal-derivedcomponents with non-animal-derived and/or recombinant components, inpart because non-animal-derived and/or recombinant components can beproduced with a higher degree of consistency than animal-derivedcomponents, and in part because non-animal-derived and/or recombinantcomponents carry less risk of contamination with toxic and/or pathogenicsubstances than do animal-derived components. Certain embodiments aretherefore directed to a protein that is non-animal-derived and/orrecombinant. Other embodiments are directed to a medium, wherein some orall of the components of the medium are non-animal-derived and/orrecombinant. In one embodiment, the protein is recombinant serumalbumin. In another embodiment, the protein is recombinant human serumalbumin. In yet another embodiment, the protein is recombinant serumalbumin and all of the components of the medium are non-animal-derivedand/or recombinant.

The N-terminus of serum albumin can contain a nickel- and copper-bindingdomain, which may be an important antigenic determinant. Deleting theaspartic acid residue from the N-terminus of serum albumin can eliminatethe nickel- and copper-binding activity of serum albumin, and can resultin a hypoallergenic variant of the protein. Certain embodiments aretherefore directed to a protein that has modified bindingcharacteristics and/or other desirable characteristics such ashypoallergenicity. In one embodiment, the protein is serum albumin, andthe serum albumin lacks an N-terminal aspartic acid.

Other embodiments are directed to a method for transfecting a cell. Inone embodiment, a cell is transfected with one or more nucleic acids,and the transfection is performed using a transfection reagent, such asa lipid-based transfection reagent. In one embodiment, the one or morenucleic acids includes at least one RNA molecule. In another embodiment,the cell is transfected with one or more nucleic acids, and the one ormore nucleic acids encodes at least one of: p53, TERT, a cytokine, asecreted protein, a membrane-bound protein, an enzyme, a gene-editingprotein, a chromatin-modifying protein, a DNA-binding protein, atranscription factor, a histone deacetylase, a pathogen-associatedmolecular pattern, and a tumor-associated antigen or a biologicallyactive fragment, analogue, variant or family-member thereof. In anotherembodiment, the cell is transfected repeatedly, such as at least about 2times during about 10 consecutive days, or at least about 3 times duringabout 7 consecutive days, or at least about 4 times during about 6consecutive days.

Reprogramming can be performed by transfecting cells with one or morenucleic acids encoding one or more reprogramming factors, and culturingthe cells in a medium that supports the reprogrammed cells. Examples ofreprogramming factors include, but are not limited to: Oct4 protein,Sox2 protein, Klf4 protein, c-Myc protein, 1-Myc protein, TERT protein,Nanog protein, Lin28 protein, Utf1 protein, Aicda protein, miR200micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA andbiologically active fragments, analogues, variants and family-membersthereof. Certain embodiments are therefore directed to a method forreprogramming a cell. In one embodiment, the cell is reprogrammed bytransfecting the cell with one or more nucleic acids encoding one ormore reprogramming factors. In one embodiment, the one or more nucleicacids includes an RNA molecule that encodes Oct4 protein. In anotherembodiment, the one or more nucleic acids also includes one or more RNAmolecules that encodes Sox2 protein, Klf4 protein, and c-Myc protein. Inyet another embodiment, the one or more nucleic acids also includes anRNA molecule that encodes Lin28 protein. In one embodiment, the cell isa human skin cell, and the human skin cell is reprogrammed to apluripotent stem cell. In another embodiment, the cell is a human skincell, and the human skin cell is reprogrammed to a glucose-responsiveinsulin-producing cell. Examples of other cells that can be reprogrammedand other cells to which a cell can be reprogrammed include, but are notlimited to: skin cells, pluripotent stem cells, mesenchymal stem cells,β-cells, retinal pigmented epithelial cells, hematopoietic cells,cardiac cells, airway epithelial cells, neural stem cells, neurons,glial cells, bone cells, blood cells, and dental pulp stem cells. In oneembodiment, the cell is cultured in a medium that supports thereprogrammed cell. In one embodiment, the medium also supports the cell.

Importantly, infecting skin cells with viruses encoding Oct4, Sox2,Klf4, and c-Myc, combined with culturing the cells in a medium thatsupports the growth of cardiomyocytes, has been reported to causereprogramming of the skin cells to cardiomyocytes, without firstreprogramming the skin cells to pluripotent stem cells (See Efs et alNat Cell Biol. 2011;13:215-22, the contents of which are herebyincorporated by reference). In certain situations, for example whengenerating a personalized therapeutic, direct reprogramming(reprogramming one somatic cell to another somatic cell without firstreprogramming the somatic cell to a pluripotent stem cell, also known as“transdifferentiation”) may be desirable, in part because culturingpluripotent stem cells can be time-consuming and expensive, theadditional handling involved in establishing and characterizing a stablepluripotent stem cell line can carry an increased risk of contamination,and the additional time in culture associated with first producingpluripotent stem cells can carry an increased risk of genomicinstability and the acquisition of mutations, including point mutations,copy-number variations, and karyotypic abnormalities. Certainembodiments are therefore directed to a method for reprogramming asomatic cell, wherein the cell is reprogrammed to a somatic cell, andwherein a characterized pluripotent stem-cell line is not produced.

Previously reported methods for reprogramming cells by transfecting themwith RNA encoding reprogramming factors require the use of feeders. Inmany situations, the use of feeders may not be desirable, in partbecause feeders may be derived from animal or allogeneic sources, andmay thus carry risks of immunogenicity and contamination with pathogens.It has now been discovered that the medium of the present invention canenable RNA reprogramming without feeders. It has been further discoveredthat reprogramming cells according to the methods of the presentinvention, wherein the cells are not contacted with feeders, can berapid, efficient, and reliable. Certain embodiments are thereforedirected to a method for reprogramming a cell, wherein the cell is notcontacted with feeders.

It has now been discovered that reprogramming efficiency can correlatewith starting cell density when cells are reprogrammed according to themethods of the present invention. Certain embodiments are thereforedirected to a method for reprogramming cells, wherein the cells areplated at a density of between about 100 cells/cm² and about 100,000cells/cm². In one embodiment, the cells are plated at a density ofbetween about 100 cells/cm² and about 10,000 cells/cm² or between about2000 cells/cm² and about 20,000 cells/cm², or between about 1000cells/cm² and about 2000 cells/cm².

It has been further discovered that, in certain situations, fewer totaltransfections may be required to reprogram a cell according to themethods of the present invention than according to other methods.Certain embodiments are therefore directed to a method for reprogramminga cell, wherein between about 2 and about 12 transfections are performedduring about 20 consecutive days, or between about 4 and about 10transfections are performed during about 15 consecutive days, or betweenabout 4 and about 8 transfections are performed during about 10consecutive days. It is recognized that when nucleic acids are added toa medium in which a cell is cultured, the cell may likely come intocontact with and/or internalize more than one nucleic acid moleculeeither simultaneously or at different times. A cell can therefore becontacted with a nucleic acid more than once, e.g. repeatedly, even whennucleic acids are added only once to a medium in which the cell iscultured.

Feeders can promote adhesion of cells to a surface by secretingmolecules such as collagen that bind to the surface (“cell-adhesionmolecules”). Proteins, including integrins, on the surface of cells canbind to these cell-adhesion molecules, which can result in the cellsadhering to the surface. It has now been discovered that cells can bereprogrammed, including without feeders, by coating a surface with oneor more cell-adhesion molecules. It has been further discovered that thecell-adhesion molecules fibronectin and vitronectin are particularlywell suited for this purpose. Certain embodiments are therefore directedto a method for transfecting, reprogramming, and/or gene-editing a cell,wherein the cell is contacted with a surface that is contacted with oneor more cell-adhesion molecules. In one embodiment, the one or morecell-adhesion molecules includes at least one of: poly-L-lysine,poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, andlaminin or a biologically active fragment, analogue, variant orfamily-member thereof. In another embodiment, the one or morecell-adhesion molecules is fibronectin or a biologically active fragmentthereof. In yet another embodiment, the fibronectin is recombinant. Instill another embodiment, the one or more cell-adhesion molecules is amixture of fibronectin and vitronectin or biologically active fragmentsthereof. In another embodiment, the fibronectin and vitronectin are eachpresent at a concentration of about 100 ng/cm² on the surface and/or ata concentration of about lug/mL in a solution used to coat the surface.In a still another embodiment, both the fibronectin and vitronectin arerecombinant. Contacting of the surface with the one or morecell-adhesion molecules can be performed as an independent step, and/orby including the one or more cell-adhesion molecules in the medium.

Of note, nucleic acids can contain one or more non-canonical, or“modified”, residues (e.g. a residue other than adenine, guanine,thymine, uracil, and cytosine or the standard nucleoside, nucleotide,deoxynucleoside or deoxynucleotide derivatives thereof). Of particularnote, pseudouridine-5′-triphosphate can be substituted foruridine-5′-triphosphate in an in vitro-transcription reaction to yieldsynthetic RNA, wherein up to 100% of the uridine residues of thesynthetic RNA may be replaced with pseudouridine residues. Invitro-transcription can yield RNA with residual immunogenicity, evenwhen pseudouridine and 5-methylcytidine are completely substituted foruridine and cytidine, respectively (See Angel Reprogramming HumanSomatic Cells to Pluripotency Using RNA [Doctoral Thesis]. Cambridge,Mass.: MIT; 2011, the contents of which are hereby incorporated byreference). For this reason, it is common to add an immunosuppressant tothe transfection medium when transfecting cells with RNA. In certainsituations, adding an immunosuppressant to the transfection medium maynot be desirable, in part because the recombinant immunosuppressant mostcommonly used for this purpose, B18R, can be expensive and difficult tomanufacture. It has now been discovered that cells can be transfectedand/or reprogrammed according to the methods of the present invention,without using B18R or any other immunosuppressant. It has been furtherdiscovered that reprogramming cells according to the methods of thepresent invention without using immunosuppressants can be rapid,efficient, and reliable. Certain embodiments are therefore directed to amethod for transfecting a cell, wherein the transfection medium does notcontain an immunosuppressant. Other embodiments are directed to a methodfor reprogramming a cell, wherein the transfection medium does notcontain an immunosuppressant. In certain situations, for example whenusing a high cell density, it may be beneficial to add animmunosuppressant to the transfection medium. Certain embodiments aretherefore directed to a method for transfecting a cell, wherein thetransfection medium contains an immunosuppressant. Other embodiments aredirected to a method for reprogramming a cell, wherein the transfectionmedium contains an immunosuppressant. In one embodiment, theimmunosuppressant is B18R or a biologically active fragment, analogue,variant or family-member thereof or dexamethasone or a derivativethereof. In one embodiment, cells are plated at a density of less thanabout 20,000 cells/cm², and the transfection medium does not contain animmunosuppressant. In another embodiment, the transfection medium doesnot contain an immunosuppressant, and the nucleic-acid dose is chosen toprevent excessive toxicity. In still another embodiment, thenucleic-acid dose is less than 2 μg/well of a 6-well plate, such asabout 0.25 μg/well of a 6-well plate or about lug/well of a 6-wellplate.

Reprogrammed cells produced according to certain embodiments of thepresent invention are suitable for therapeutic applications, includingtransplantation into patients, as they do not contain exogenous DNAsequences, and they are not exposed to animal-derived or human-derivedproducts, which may be undefined, and which may contain toxic and/orpathogenic contaminants. Furthermore, the high speed, efficiency, andreliability of certain embodiments of the present invention may reducethe risk of acquisition and accumulation of mutations and otherchromosomal abnormalities. Certain embodiments of the present inventioncan thus be used to generate cells that have a safety profile adequatefor use in therapeutic applications. For example, reprogramming cellsusing RNA and the medium of the present invention, wherein the mediumdoes not contain animal or human-derived components, can yield cellsthat have not been exposed to allogeneic material. Certain embodimentsare therefore directed to a reprogrammed cell that has a desirablesafety profile. In one embodiment, the reprogrammed cell has a normalkaryotype. In another embodiment, the reprogrammed cell has fewer thanabout 5 copy-number variations (CNVs) relative to the patient genome,such as fewer than about 3 copy-number variations relative to thepatient genome, or no copy-number variations relative to the patientgenome. In yet another embodiment, the reprogrammed cell has a normalkaryotype and fewer than about 100 single nucleotide variants in codingregions relative to the patient genome, or fewer than about 50 singlenucleotide variants in coding regions relative to the patient genome, orfewer than about 10 single nucleotide variants in coding regionsrelative to the patient genome.

Endotoxins and nucleases can co-purify and/or become associated withother proteins, such as serum albumin. Recombinant proteins, inparticular, can often have high levels of associated endotoxins andnucleases, due in part to the lysis of cells that can take place duringtheir production. Endotoxins and nucleases can be reduced, removed,replaced or otherwise inactivated by many of the methods of the presentinvention, including, for example, by acetylation, by addition of astabilizer such as sodium octanoate, followed by heat treatment, by theaddition of nuclease inhibitors to the albumin solution and/or medium,by crystallization, by contacting with one or more ion-exchange resins,by contacting with charcoal, by preparative electrophoresis or byaffinity chromatography. It has now been discovered that partially orcompletely reducing, removing, replacing or otherwise inactivatingendotoxins and/or nucleases from a medium and/or from one or morecomponents of a medium can increase the efficiency with which cells canbe transfected and reprogrammed. Certain embodiments are thereforedirected to a method for transfecting a cell with one or more nucleicacids, wherein the transfection medium is treated to partially orcompletely reduce, remove, replace or otherwise inactivate one or moreendotoxins and/or nucleases. Other embodiments are directed to a mediumthat causes minimal degradation of nucleic acids. In one embodiment, themedium contains less than about 1 EU/mL, or less than about 0.1 EU/mL,or less than about 0.01 EU/mL.

In certain situations, protein-based lipid carriers such as serumalbumin can be replaced with non-protein-based lipid carriers such asmethyl-beta-cyclodextrin. The medium of the present invention can alsobe used without a lipid carrier, for example, when transfection isperformed using a method that may not require or may not benefit fromthe presence of a lipid carrier, for example, using one or morepolymer-based transfection reagents or peptide-based transfectionreagents.

Many protein-associated molecules, such as metals, can be highly toxicto cells. This toxicity can cause decreased viability in culture, aswell as the acquisition of mutations. Certain embodiments thus have theadditional benefit of producing cells that are free from toxicmolecules.

The associated-molecule component of a protein can be measured bysuspending the protein in solution and measuring the conductivity of thesolution. Certain embodiments are therefore directed to a medium thatcontains a protein, wherein about a 10% solution of the protein in waterhas a conductivity of less than about 500 μmho/cm. In one embodiment,the solution has a conductivity of less than about 50 μmho/cm.

A low-oxygen environment can be beneficial for the culture of many typesof cells. Certain embodiments are therefore directed to a method forculturing, transfecting, reprogramming, and/or gene-editing cells,wherein the cells are cultured, transfected, reprogrammed, and/orgene-edited in a low-oxygen environment. In one embodiment, thelow-oxygen environment contains between about 2% and about 10% oxygen,or between about 4% and about 6% oxygen.

The amount of nucleic acid delivered to cells can be increased toincrease the desired effect of the nucleic acid. However, increasing theamount of nucleic acid delivered to cells beyond a certain point cancause a decrease in the viability of the cells, due in part to toxicityof the transfection reagent. It has now been discovered that when anucleic acid is delivered to a population of cells in a fixed volume(for example, cells in a region of tissue or cells grown in acell-culture vessel), the amount of nucleic acid delivered to each cellcan depend on the total amount of nucleic acid delivered to thepopulation of cells and to the density of the cells, with a higher celldensity resulting in less nucleic acid being delivered to each cell. Incertain embodiments, a cell is transfected with one or more nucleicacids more than once. Under certain conditions, for example when thecells are proliferating, the cell density may change from onetransfection to the next. Certain embodiments are therefore directed toa method for transfecting a cell with a nucleic acid, wherein the cellis transfected more than once, and wherein the amount of nucleic aciddelivered to the cell is different for two of the transfections. In oneembodiment, the cell proliferates between two of the transfections, andthe amount of nucleic acid delivered to the cell is greater for thesecond of the two transfections than for the first of the twotransfections. In another embodiment, the cell is transfected more thantwice, and the amount of nucleic acid delivered to the cell is greaterfor the second of three transfections than for the first of the samethree transfections, and the amount of nucleic acid delivered to thecells is greater for the third of the same three transfections than forthe second of the same three transfections. In yet another embodiment,the cell is transfected more than once, and the maximum amount ofnucleic acid delivered to the cell during each transfection issufficiently low to yield at least about 80% viability for at least twoconsecutive transfections.

It has now been discovered that modulating the amount of nucleic aciddelivered to a population of proliferating cells in a series oftransfections can result in both an increased effect of the nucleic acidand increased viability of the cells. It has been further discoveredthat, in certain situations, when cells are contacted with one or morenucleic acids encoding one or more reprogramming factors in a series oftransfections, the efficiency of reprogramming can be increased when theamount of nucleic acid delivered in later transfections is greater thanthe amount of nucleic acid delivered in earlier transfections, for atleast part of the series of transfections. Certain embodiments aretherefore directed to a method for reprogramming a cell, wherein one ormore nucleic acids is repeatedly delivered to the cell in a series oftransfections, and the amount of the nucleic acid delivered to the cellis greater for at least one later transfection than for at least oneearlier transfection. In one embodiment, the cell is transfected betweenabout 2 and about 10 times, or between about 3 and about 8 times, orbetween about 4 and about 6 times. In another embodiment, the one ormore nucleic acids includes at least one RNA molecule, the cell istransfected between about 2 and about 10 times, and the amount ofnucleic acid delivered to the cell in each transfection is the same asor greater than the amount of nucleic acid delivered to the cell in themost recent previous transfection. In yet another embodiment, the amountof nucleic acid delivered to the cell in the first transfection isbetween about 20 ng/cm² and about 250 ng/cm², or between 100 ng/cm² and600 ng/cm². In yet another embodiment, the cell is transfected about 5times at intervals of between about 12 and about 48 hours, and theamount of nucleic acid delivered to the cell is about 25 ng/cm² for thefirst transfection, about 50 ng/cm² for the second transfection, about100 ng/cm² for the third transfection, about 200 ng/cm² for the fourthtransfection, and about 400 ng/cm² for the fifth transfection. In yetanother embodiment, the cell is further transfected at least once afterthe fifth transfection, and the amount of nucleic acid delivered to thecell is about 400 ng/cm².

Certain embodiments are directed to a method for transfecting a cellwith a nucleic acid, wherein the amount of nucleic acid is determined bymeasuring the cell density, and choosing the amount of nucleic acid totransfect based on the measurement of cell density. In one embodiment,the cell is present in an in vitro culture, and the cell density ismeasured by optical means. In another embodiment, the cell istransfected repeatedly, the cell density increases between twotransfections, and the amount of nucleic acid transfected is greater forthe second of the two transfections than for the first of the twotransfections.

It has now been discovered that, in certain situations, the transfectionefficiency and viability of cells cultured in the medium of the presentinvention can be improved by conditioning the medium. Certainembodiments are therefore directed to a method for conditioning amedium. Other embodiments are directed to a medium that is conditioned.In one embodiment, the feeders are fibroblasts, and the medium isconditioned for approximately 24 hours. Other embodiments are directedto a method for transfecting a cell, wherein the transfection medium isconditioned. Other embodiments are directed to a method forreprogramming and/or gene-editing a cell, wherein the medium isconditioned. In one embodiment, the feeders are mitotically inactivated,for example, by exposure to a chemical such as mitomycin-C or byexposure to gamma radiation. In certain embodiments, it may bebeneficial to use only autologous materials, in part to, for example andnot wishing to be bound by theory, avoid the risk of diseasetransmission from the feeders to the cell. Certain embodiments aretherefore directed to a method for transfecting a cell, wherein thetransfection medium is conditioned, and wherein the feeders are derivedfrom the same individual as the cell being transfected. Otherembodiments are directed to a method for reprogramming and/orgene-editing a cell, wherein the medium is conditioned, and wherein thefeeders are derived from the same individual as the cell beingreprogrammed and/or gene-edited.

Several molecules can be added to media by conditioning. Certainembodiments are therefore directed to a medium that is supplemented withone or more molecules that are present in a conditioned medium. In oneembodiment, the medium is supplemented with Wnt1, Wnt2, Wnt3, Wnt3a or abiologically active fragment, analogue, variant, agonist, orfamily-member thereof. In another embodiment, the medium is supplementedwith TGF-β or a biologically active fragment, analogue, variant,agonist, or family-member thereof. In yet another embodiment, a cell isreprogrammed according to the method of the present invention, whereinthe medium is not supplemented with TGF-β for between about 1 and about5 days, and is then supplemented with TGF-β for at least about 2 days.In yet another embodiment, the medium is supplemented with IL-6, IL-6Ror a biologically active fragment, analogue, variant, agonist, orfamily-member thereof. In yet another embodiment, the medium issupplemented with a sphingolipid or a fatty acid. In still anotherembodiment, the sphingolipid is lysophosphatidic acid,lysosphingomyelin, sphingosine-1-phosphate or a biologically activeanalogue, variant or derivative thereof.

In addition to mitotically inactivating cells, under certain conditions,irradiation can change the gene expression of cells, causing cells toproduce less of certain proteins and more of certain other proteins thatnon-irradiated cells, for example, members of the Wnt family ofproteins. In addition, certain members of the Wnt family of proteins canpromote the growth and transformation of cells. It has now beendiscovered that, in certain situations, the efficiency of RNAreprogramming can be greatly increased by contacting the cell with amedium that is conditioned using irradiated feeders instead ofmitomycin-c-treated feeders. It has been further discovered that theincrease in reprogramming efficiency observed when using irradiatedfeeders is caused in part by Wnt proteins that are secreted by thefeeders. Certain embodiments are therefore directed to a method forreprogramming a cell, wherein the cell is contacted with Wnt1, Wnt2,Wnt3, Wnt3a or a biologically active fragment, analogue, variant,family-member or agonist thereof, including agonists of downstreamtargets of Wnt proteins, and/or agents that mimic one or more of thebiological effects of Wnt proteins, for example,2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyepyrimidine.

It has now been discovered that the medium of the present invention canbe used to maintain cells, including fibroblasts and human pluripotentstem cells, in culture (i.e. as a “maintenance medium”). Certainembodiments are therefore directed to a medium that is used as amaintenance medium. In one embodiment, the medium does not contain anyhuman-derived components. In another embodiment, the medium ischemically defined.

Because of the low efficiency of many DNA-based reprogramming methods,these methods may be difficult or impossible to use with cells derivedfrom patient samples, which may contain only a small number of cells. Incontrast, the high efficiency of certain embodiments of the presentinvention can allow reliable reprogramming from small numbers of cells,including from single cells. Certain embodiments can thus be used toreprogram cells from a biopsy sample, including without firstestablishing a large culture. Reprogramming cells directly from a biopsymay be desirable in certain situations, for example when generating apersonalized therapeutic, in part because establishing a large cultureof primary cells can be time-consuming, the additional handling involvedin establishing a large culture can carry an increased risk ofcontamination, and the additional time in culture can carry an increasedrisk of genomic instability and the acquisition of mutations, includingpoint mutations, copy-number variations, and karyotypic abnormalities.Certain embodiments are therefore directed to a method for reprogramminga cell by first harvesting the cell from a patient or from a biopsysample, and then reprogramming the cell. In one embodiment, the cell isreprogrammed without first establishing a large culture, preferably afirst transfection is performed before the culture is passaged more thantwice. In another embodiment, the cell is harvested from a patient, anda first transfection is performed after no more than about 14 days fromthe time the cell is first plated. In yet another embodiment, the cellis harvested from a biopsy sample, and a first transfection is performedafter no more than about 7 days from the time the cell is first plated.In yet another embodiment, the biopsy is a full-thickness dermal punchbiopsy, the cell is harvested from the biopsy sample by treatment withone or more enzymes, the cell is plated on a surface that is coated withone or more cell-adhesion molecules and/or the cell is plated in amedium that contains a cell-adhesion molecule, the cell is transfectedwith one or more nucleic acids comprising at least one RNA molecule, anda first transfection is performed after no more than about 14 days fromthe time the cell is first plated. In still another embodiment, theenzyme is collagenase. In yet another embodiment, the collagenase isanimal-component free. In another embodiment, the collagenase is presentat a concentration of between about 0.1 mg/mL and about 10 mg/mL, orbetween about 0.5 mg/mL and about 5 mg/mL. In yet another embodiment,the cell is harvested from blood. In yet another embodiment, the cell isplated in a medium containing one or more proteins that is derived fromthe patient's blood. In still another embodiment, the cell is plated inDMEM/F12+2mM L-alanyl-L-glutamine+between about 5% and about 25%patient-derived serum, or between about 10% and about 20%patient-derived serum, or about 20% patient-derived serum.

It has now been discovered that, in certain situations, transfectingcells with a mixture of RNA encoding Oct4, Sox2, Klf4, and c-Myc usingthe medium of the present invention can cause the rate of proliferationof the cells to increase. When the amount of RNA delivered to the cellsis too low to ensure that all of the cells are transfected, only afraction of the cells may show an increased proliferation rate. Incertain situations, such as when generating a personalized therapeutic,increasing the proliferation rate of cells may be desirable, in partbecause doing so can reduce the time necessary to generate thetherapeutic, and therefore can reduce the cost of the therapeutic.Certain embodiments are therefore directed to a method for transfectinga cell with a mixture of RNA encoding Oct4, Sox2, Klf4, and c-Myc,wherein the cell exhibits an increased proliferation rate. In oneembodiment, cells showing an increased proliferation rate are isolatedfrom the culture. In another embodiment, cells showing an increasedproliferation rate are expanded and cultured in a medium that supportsthe growth of one or more cell types, and are reprogrammed to a cell ofone of the one or more cell types.

Many diseases are associated with one or more mutations. Mutations canbe corrected by contacting a cell with a nucleic acid that encodes aprotein that, either alone or in combination with other molecules,corrects the mutation (an example of gene-editing). Examples of suchproteins include: zinc finger nucleases and TALENs. Certain embodimentsare therefore directed to a method for transfecting a cell with anucleic acid, wherein the nucleic acid encodes a protein that, eitheralone or in combination with other molecules, creates a single-strand ordouble-strand break in a DNA molecule. In a one embodiment, the proteinis a zinc finger nuclease or a TALEN. In another embodiment, the nucleicacid is an RNA molecule. In yet another embodiment, the single-strand ordouble-strand break is within about 5,000,000 bases of the transcriptionstart site of a gene selected from the group: CCR5, CXCR4, GAD1, GAD2,CFTR, HBA1, HBA2, HBB, HBD, FANCA, XPA, XPB, XPC, ERCC2, POLH, HTT, DMD,SOD1, APOE, PRNP, BRCA1, and BRCA2 or an analogue, variant orfamily-member thereof. In yet another embodiment, the cell istransfected with a nucleic acid that acts as a repair template by eithercausing the insertion of a DNA sequence in the region of thesingle-strand or double-strand break or by causing the DNA sequence inthe region of the single-strand or double-strand break to otherwisechange. In yet another embodiment, the cell is reprogrammed, andsubsequently, the cell is gene-edited. In yet another embodiment, thecell is gene-edited, and subsequently, the cell is reprogrammed. In yetanother embodiment, the gene-editing and reprogramming are performedwithin about 7 days of each other. In yet another embodiment, thegene-editing and reprogramming occur simultaneously or on the same day.In yet another embodiment, the cell is a skin cell, the skin cell isgene-edited to disrupt the CCR5 gene, the skin cell is reprogrammed to ahematopoietic stem cell, thus producing a therapeutic for HIV/AIDS, andthe therapeutic is introduced into a patient with HIV/AIDS. In yetanother embodiment, the skin cell is derived from the same patient intowhom the therapeutic is introduced.

Genes that can be edited according to the methods of the presentinvention to produce therapeutics of the present invention include genesthat can be edited to restore normal function, as well as genes that canbe edited to reduce or eliminate function. Such genes include, but arenot limited to beta globin (HBB), mutations in which can cause sicklecell disease (SCD) and β-thalassemia, breast cancer 1, early onset(BRCA1) and breast cancer 2, early onset (BRCA2), mutations in which canincrease susceptibility to breast cancer, C-C chemokine receptor type 5(CCR5) and C-X-C chemokine receptor type 4 (CXCR4), mutations in whichcan confer resistance to HIV infection, cystic fibrosis transmembraneconductance regulator (CFTR), mutations in which can cause cysticfibrosis, dystrophin (DMD), mutations in which can cause musculardystrophy, including Duchenne muscular dystrophy and Becker's musculardystrophy, glutamate decarboxylase 1 and glutamate decarboxylase 2(GAD1, GAD2), mutations in which can prevent autoimmune destruction ofβ-cells, hemoglobin alpha 1, hemoglobin alpha 2, and hemoglobin delta(HBA1, HBA2, and HBD), mutations in which can cause thalassemia,Huntington (HTT), mutations in which can cause Huntington's disease,superoxide dismutase 1 (SOD1), mutations in which can cause amyotrophiclateral sclerosis (ALS), XPA, XPB, XPC, XPD (ERCC6) and polymerase (DNAdirected), eta (POLH), mutations in which can cause xerodermapigmentosum, leucine-rich repeat kinase 2 (LRRK2), mutations in whichcan cause Parkinson's disease, and Fanconi anemia, complementationgroups A, B, C, D1, D2, E, F, G, I, J, L, M, N, P (FANCA, FANCB, FANCC,FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN,FANCP), and RAD51 homolog C (S. cerevisiae) (RAD51C), mutations in whichcan cause Fanconi anemia.

Certain embodiments are directed to a therapeutic comprising a nucleicacid that encodes one or more gene-editing proteins. Other embodimentsare directed to a therapeutic comprising one or more cells that aretransfected, reprogrammed, and/or gene-edited according to the methodsof the present invention. In one embodiment, a cell is transfected,reprogrammed, and/or gene-edited, and the transfected, reprogrammed,and/or gene-edited cell is introduced into a patient. In anotherembodiment, the cell is harvested from the same patient into whom thetransfected, reprogrammed and/or gene-edited cell is introduced.Examples of diseases that can be treated with therapeutics of thepresent invention include, but are not limited to Alzheimer's disease,spinal cord injury, amyotrophic lateral sclerosis, cystic fibrosis,heart disease, including ischemic and dilated cardiomyopathy, maculardegeneration, Parkinson's disease, Huntington's disease, diabetes,sickle-cell anemia, thalassemia, Fanconi anemia, xeroderma pigmentosum,muscular dystrophy, severe combined immunodeficiency, hereditary sensoryneuropathy, cancer, and HIV/AIDS. In certain embodiments, thetherapeutic comprises a cosmetic. In one embodiment, a cell is harvestedfrom a patient, the cell is reprogrammed and expanded to a large numberof adipose cells, thus producing a cosmetic, and the cosmetic isintroduced into the patient. In still another embodiment, the cosmeticis used for tissue reconstruction.

While detailed examples are provided herein for the production ofspecific types of cells and for the production of therapeuticscomprising specific types of cells, it is recognized that the methods ofthe present invention can be used to produce many other types of cells,and to produce therapeutics comprising one or more of many other typesof cells, for example, by reprogramming a cell according to the methodsof the present invention, and culturing the cell under conditions thatmimic one or more aspects of development by providing conditions thatresemble the conditions present in the cellular microenvironment duringdevelopment.

Certain embodiments are directed to a library of cells with a variety ofhuman leukocyte antigen (HLA) types (“HLA-matched libraries”). AnHLA-matched library may be beneficial in part because it can provide forthe rapid production and/or distribution of therapeutics without thepatient having to wait for a therapeutic to be produced from thepatient's cells. Such a library may be particularly beneficial for thetreatment of heart disease and diseases of the blood and/or immunesystem for which patients may benefit from the immediate availability ofa therapeutic.

Certain embodiments are directed to a cell that is used for tissue/organmodeling and/or disease modeling. In one embodiment, a skin cell isreprogrammed and expanded to a large number of cardiac cells, and thecardiac cells are used for screening bioactive molecules forcardiotoxicity (an example of safety testing). In another embodiment, askin cell from a patient with Alzheimer's disease is reprogrammed andexpanded to a large number of cortical neurons, and the cortical neuronsare used for screening bioactive molecules for reducing the accumulationof insoluble plaques (an example of efficacy testing). Certainembodiments of the present invention are therefore useful for safetytesting and/or efficacy testing.

Certain embodiments are directed to a method for encapsulating cellsand/or seeding cells in a scaffold, and to cells that are encapsulatedand/or cells that are seeded in a scaffold. In certain situations,encapsulating cells may be beneficial, in part because encapsulatedcells may be less immunogenic than non-encapsulated cells. In oneembodiment, a cell is reprogrammed to a glucose-responsiveinsulin-producing cell, the glucose-responsive insulin-producing cell isencapsulated in a material such as alginate, and the encapsulatedglucose-responsive insulin-producing cell is introduced into a patientwith type 1 diabetes. In another embodiment, the introducing is byintraperitoneal injection or intraportal injection. In certainsituations, seeding cells in a scaffold may be beneficial, in partbecause a scaffold can provide mechanical stability. In one embodiment,a cell is reprogrammed and expanded into a large number of fibroblastsand keratinocytes, the fibroblasts and keratinocytes are seeded in ascaffold comprising collagen, and the seeded scaffold is applied to awound, forming a synthetic skin graft. In another embodiment, a cell isreprogrammed, the reprogrammed cell is mixed with a scaffold in liquidor slurry form, the mixture is introduced into the patient, and thestiffness of the scaffold increases upon or after introduction.

Certain embodiments are directed to a method for purifying cells.Transfecting, reprogramming, and gene-editing can often producepopulations of cells that include cells with the desired phenotype andcells with one or more undesired phenotypes. Certain embodiments aretherefore directed to a method for purifying transfected, reprogrammed,and/or gene-edited cells. In one embodiment, the cells are purifiedusing a density gradient. In another embodiment, the cells are purifiedby contacting the cells with one or more antibodies that allows theseparation of cells having one or more desired phenotypes from cellshaving one or more undesired phenotypes. In another embodiment, theantibody is bound to a substrate, preferably a magnetic bead. In yetanother embodiment, the antibody is bound to a fluorescent molecule, andthe separation is performed by fluorescence activated cell sorting(FACS) or other similar means. In another embodiment, cells with anundesired phenotype are prevented from proliferating, preferably bycontacting the cells with one or more molecules that prevents the cellsfrom dividing, preferably mitomycin-c, 5-aza-deoxycytidine, fluorouracilor a biologically active analogue or derivative thereof. Otherembodiments are directed to a therapeutic comprising cells that arepurified to enrich the fraction of cells having one or more desiredphenotypes.

Certain embodiments are directed to a method for producing animalmodels, including models of mutations and diseases. In one embodiment,an animal skin cell is gene-edited and reprogrammed to a pluripotentstem cell. In another embodiment, about 1-100 reprogrammed andgene-edited cells are injected into a blastocyst, and the blastocyst isimplanted into the uterine horn of an animal. In one embodiment, theanimal is selected from the group: a cat, a dog, a mouse, a pig, ahorse, a cow, a chicken, a sheep, a goat, a fish, a primate, and a rat.In another embodiment, the animal is a rat.

Certain non-canonical nucleotides, when incorporated into synthetic RNAmolecules, can reduce the toxicity of the synthetic RNA molecules, inpart by interfering with binding of proteins that detect exogenousnucleic acids, for example, protein kinase R, Rig-1 and theoligoadenylate synthetase family of proteins. Non-canonical nucleotidesthat have been reported to reduce the toxicity of synthetic RNAmolecules when incorporated therein include: pseudouridine,5-methyluridine, 2-thiouridine, 5-methylcytidine, N6-methyladenosine,and certain combinations thereof. However, the chemical characteristicsof non-canonical nucleotides that can enable them to lower the toxicityof synthetic RNA molecules have, until this point, remained unknown.Furthermore, incorporation of large amounts of most non-canonicalnucleotides, for example, 5-methyluridine, 2-thiouridine,5-methylcytidine, and N6-methyladenosine, can reduce the efficiency withwhich synthetic RNA molecules can be translated into protein, limitingthe utility of synthetic RNA molecules containing these nucleotides inapplications that require protein expression. In addition, whilepseudouridine can be completely substituted for uridine in synthetic RNAmolecules without reducing the efficiency with which the synthetic RNAmolecules can be translated into protein, in certain situations, forexample, when performing frequent, repeated transfections, synthetic RNAmolecules containing only adenosine, guanosine, cytidine, andpseudouridine can exhibit excessive toxicity.

It has now been discovered that synthetic RNA molecules containing oneor more non-canonical nucleotides that include one or more substitutionsat the 2C and/or 4C and/or 5C positions in the case of a pyrimidine orthe 6C and/or 7N and/or 8C positions in the case of a purine can be lesstoxic than synthetic RNA molecules containing only canonicalnucleotides, due in part to the ability of substitutions at thesepositions to interfere with recognition of synthetic RNA molecules byproteins that detect exogenous nucleic acids, and furthermore, thatsubstitutions at these positions can have minimal impact on theefficiency with which the synthetic RNA molecules can be translated intoprotein, due in part to the lack of interference of substitutions atthese positions with base-pairing and base-stacking interactions.

Examples of non-canonical nucleotides that include one or moresubstitutions at the 2C and/or 4C and/or 5C positions in the case of apyrimidine or the 6C and/or 7N and/or 8C positions in the case of apurine include, but are not limited to: 2-thiouridine, 5-azauridine,pseudouridine, 4-thiouridine, 5-methyluridine, 5-aminouridine,5-hydroxyuridine, 5-methyl-5-azauridine, 5-amino-5-azauridine,5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine,5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine,4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine,4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine,4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine,4-thio-5-aminopseudoinidine, 4-thio-5-hydroxypseudouridine,2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine,N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine,5-hydroxycytidine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine,5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine,5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine,N4-methyl-5-azacytidine, N4-methylpseudoisocytidine,2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine,2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine,2-thio-5-methylcytidine, 2-thio-5-aminocytidine,2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine,2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine,2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine,2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine,2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine,N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine,N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine,N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine,N4-methyl-5-aminopseudoisocytidine,N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine,N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine,N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine,N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine,N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine,N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine,N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine,N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine,N4-hydroxy-5-hydroxy-5-azacytidine,N4-hydroxy-5-methylpseudoisocytidine,N4-hydroxy-5-aminopseudoisocytidine,N4-hydroxy-5-hydroxypseudoisocytidine,2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine,2-thio-N4-methyl-5-hydroxycytidine,2-thio-N4-methyl-5-methyl-5-azacytidine,2-thio-N4-methyl-5-amino-5-azacytidine,2-thio-N4-methyl-5-hydroxy-5-azacytidine,2-thio-N4-methyl-5-methylpseudoisocytidine,2-thio-N4-methyl-5-aminopseudoisocytidine,2-thio-N4-methyl-5-hydroxypseudoisocytidine,2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine,2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine,2-thio-N4-amino-5-hydroxycytidine,2-thio-N4-amino-5-methyl-5-azacytidine,2-thio-N4-amino-5-amino-5-azacytidine,2-thio-N4-amino-5-hydroxy-5-azacytidine,2-thio-N4-amino-5-methylpseudoisocytidine,2-thio-N4-amino-5-aminopseudoisocytidine,2-thio-N4-amino-5-hydroxypseudoisocytidine,2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine,2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine,2-thio-N4-hydroxy-5-hydroxycytidine,2-thio-N4-hydroxy-5-methyl-5-azacytidine,2-thio-N4-hydroxy-5-amino-5-azacytidine,2-thio-N4-hydroxy-5-hydroxy-5-azacytidine,2-thio-N4-hydroxy-5-methylpseudoisocytidine,2-thio-N4-hydroxy-5-aminopseudoisocytidine,2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine,N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine,8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine,7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine,N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine,N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine,N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine,N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine,8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine,7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine. Note thatalternative naming schemes exist for certain non-canonical nucleotides.For example, in certain situations, 5-methylpseudouridine can bereferred to as “3-methylpseudouridine” or “N3-methylpseudouridine”.

Nucleotides that contain the prefix “amino” can refer to any nucleotidethat contains a nitrogen atom bound to the atom at the stated positionof the nucleotide, for example, 5-aminocytidine can refer to5-aminocytidine, 5-methylaminocytidine, and 5-nitrocytidine. Similarly,nucleotides that contain the prefix “methyl” can refer to any nucleotidethat contains a carbon atom bound to the atom at the stated position ofthe nucleotide, for example, 5-methylcytidine can refer to5-methylcytidine, 5-ethylcytidine, and 5-hydroxymethylcytidine,nucleotides that contain the prefix “thio” can refer to any nucleotidethat contains a sulfur atom bound to the atom at the given position ofthe nucleotide, and nucleotides that contain the prefix “hydroxy” canrefer to any nucleotide that contains an oxygen atom bound to the atomat the given position of the nucleotide.

Certain embodiments are therefore directed to a synthetic RNA molecule,wherein the synthetic RNA molecule contains one or more nucleotides thatincludes one or more substitutions at the 2C and/or 4C and/or 5Cpositions in the case of a pyrimidine or the 6C and/or 7N and/or 8Cpositions in the case of a purine. Other embodiments are directed to atherapeutic, wherein the therapeutic contains one or more synthetic RNAmolecules, and wherein the one or more synthetic RNA molecules containsone or more nucleotides that includes one or more substitutions at the2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6Cand/or 7N and/or 8C positions in the case of a purine. In oneembodiment, the therapeutic comprises a transfection reagent. In anotherembodiment, the transfection reagent comprises a cationic lipid,liposome or micelle. In still another embodiment, the liposome ormicelle comprises folate and the therapeutic composition has anti-canceractivity. In another embodiment, the one or more nucleotides includes atleast one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine,5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine,4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine,5-aminopseudouridine, pseudoisocytidine, N4-methylcytidine,2-thiocytidine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine,5-methylcytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine,5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine,5-methylpseudoisocytidine, 7-deazaadenosine, 7-deazaguanosine,6-thioguanosine, and 6-thio-7-deazaguanosine. In another embodiment, theone or more nucleotides includes at least one of pseudouridine,2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine,5-methyluridine, 5-aminouridine, 2-thiopseudouridine,4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, and5-aminopseudouridine and at least one of pseudoisocytidine,N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine,5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine,2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine,5-aminopseudoisocytidine, and 5-methylpseudoisocytidine. In stillanother embodiment, the one or more nucleotides include at least one ofpseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine,5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine,4-thiopseudouridine, 5-hydroxypseudouridine, and 5-methylpseudouridine,5-aminopseudouridine and at least one of pseudoisocytidine,N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine,5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine,2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine,5-aminopseudoisocytidine, and 5-methylpseudoisocytidine and at least oneof 7-deazaguanosine, 6-thioguanosine, and 6-thio-7-deazaguanosine. Inyet another embodiment, the one or more nucleotides includes:5-methylcytidine and 7-deazaguanosine. In another embodiment, the one ormore nucleotides also includes pseudouridine or 4-thiouridine or5-methyluridine or 5-aminouridine or 4-thiopseudouridine or5-methylpseudouridine or 5-aminopseudouridine. In a still anotherembodiment, the one or more nucleotides also includes 7-deazaadenosine.In another embodiment, the one or more nucleotides includes:pseudoisocytidine and 7-deazaguanosine and 4-thiouridine. In yet anotherembodiment, the one or more nucleotides includes: pseudoisocytidine or7-deazaguanosine and pseudouridine. In still another embodiment, the oneor more nucleotides includes: 5-methyluridine and 5-methylcytidine and7-deazaguanosine. In a further embodiment, the one or more nucleotidesincludes: pseudouridine or 5-methylpseudouridine and 5-methylcytidineand 7-deazaguanosine. In another embodiment, the one or more nucleotidesincludes: pseudoisocytidine and 7-deazaguanosine and pseudouridine.

Certain non-canonical nucleotides can be incorporated more efficientlythan other non-canonical nucleotides into synthetic RNA molecules by RNApolymerases that are commonly used for in vitro transcription, due inpart to the tendency of these certain non-canonical nucleotides toparticipate in standard base-pairing interactions and base-stackinginteractions, and to interact with the RNA polymerase in a mannersimilar to that in which the corresponding canonical nucleotideinteracts with the RNA polymerase. As a result, certain nucleotidemixtures containing one or more non-canonical nucleotides can bebeneficial in part because in vitro-transcription reactions containingthese nucleotide mixtures can yield a large quantity of synthetic RNA.Certain embodiments are therefore directed to a nucleotide mixturecontaining one or more nucleotides that includes one or moresubstitutions at the 2C and/or 4C and/or 5C positions in the case of apyrimidine or the 6C and/or 7N and/or 8C positions in the case of apurine. Nucleotide mixtures include, but are not limited to (numberspreceding each nucleotide indicate an exemplary fraction of thenon-canonical nucleotide triphosphate in an in vitro-transcriptionreaction, for example, 0.2 pseudoisocytidine refers to a reactioncontaining adenosine-5′-triphosphate, guanosine-5′-triphosphate,uridine-5′-triphosphate, cytidine-5′-triphosphate, andpseudoisocytidine-5′-triphosphate, whereinpseudoisocytidine-5′-triphosphate is present in the reaction at anamount approximately equal to 0.2 times the total amount ofpseudoisocytidine-5′-triphosphate+cytidine-5′-triphosphate that ispresent in the reaction, with amounts measured either on a molar or massbasis, and wherein more than one number preceding a nucleoside indicatesa range of exemplary fractions): 1.0 pseudouridine, 0.1-0.82-thiouridine, 0.1-0.8 5-methyluridine, 0.2-1.0 5-hydroxyuridine,0.1-1.0 5-aminouridine, 0.1-1.0 4-thiouridine, 0.1-1.02-thiopseudouridine, 0.1-1.0 4-thiopseudouridine, 0.1-1.05-hydroxypseudouridine, 0.2-1 5-methylpseudouridine, 0.1-1.05-aminopseudouridine, 0.2-1.0 2-thiocytidine, 0.1-0.8 pseudoisocytidine,0.2-1.0 5-methylcytidine, 0.2-1.0 5-hydroxycytidine, 0.1-1.05-aminocytidine, 0.2-1.0 N4-methylcytidine, 0.2-1.05-methylpseudoisocytidine, 0.2-1.0 5-hydroxypseudoisocytidine, 0.2-1.05-aminopseudoisocytidine, 0.2-1.0 N4-methylpseudoisocytidine, 0.2-1.02-thiopseudoisocytidine, 0.2-1.0 7-deazaguanosine, 0.2-1.06-thioguanosine, 0.2-1.0 6-thio-7-deazaguanosine, 0.2-1.08-azaguanosine, 0.2-1.0 7-deaza-8-azaguanosine, 0.2-1.06-thio-8-azaguanosine, 0.1-0.5 7-deazaadenosine, and 0.1-0.5N6-methyladenosine.

It has now been discovered that combining certain non-canonicalnucleotides can be beneficial in part because the contribution ofnon-canonical nucleotides to lowering the toxicity of synthetic RNAmolecules can be additive. Certain embodiments are therefore directed toa nucleotide mixture, wherein the nucleotide mixture contains more thanone of the non-canonical nucleotides listed above, for example, thenucleotide mixture contains both pseudoisocytidine and 7-deazaguanosineor the nucleotide mixture contains both N4-methylcytidine and7-deazaguanosine, etc. In one embodiment, the nucleotide mixturecontains more than one of the non-canonical nucleotides listed above,and each of the non-canonical nucleotides is present in the mixture atthe fraction listed above, for example, the nucleotide mixture contains0.1-0.8 pseudoisocytidine and 0.2-1.0 7-deazaguanosine or the nucleotidemixture contains 0.2-1.0 N4-methylcytidine and 0.2-1.0 7-deazaguanosine,etc.

In certain situations, for example, when it may not be necessary ordesirable to maximize the yield of an in vitro-transcription reaction,nucleotide fractions other than those given above may be used. Theexemplary fractions and ranges of fractions listed above relate tonucleotide-triphosphate solutions of typical purity (greater than 90%purity). Larger fractions of these and other nucleotides can be used byusing nucleotide-triphosphate solutions of greater purity, for example,greater than about 95% purity or greater than about 98% purity orgreater than about 99% purity or greater than about 99.5% purity, whichcan be achieved, for example, by purifying the nucleotide triphosphatesolution using existing chemical-purification technologies such ashigh-pressure liquid chromatography (HPLC) or by other means. In oneembodiment, nucleotides with multiple isomers are purified to enrich thedesired isomer.

Other embodiments are directed to a method for inducing a cell toexpress a protein of interest by contacting the cell with a syntheticRNA molecule that contains one or more non-canonical nucleotides thatincludes one or more substitutions at the 2C and/or 4C and/or 5Cpositions in the case of a pyrimidine or the 6C and/or 7N and/or 8Cpositions in the case of a purine. Still other embodiments are directedto a method for transfecting, reprogramming, and/or gene-editing a cellby contacting the cell with a synthetic RNA molecule that contains oneor more non-canonical nucleotides that includes one or moresubstitutions at the 2C and/or 4C and/or 5C positions in the case of apyrimidine or the 6C and/or 7N and/or 8C positions in the case of apurine. In one embodiment, the synthetic RNA molecule is produced by invitro transcription. In one embodiment, the synthetic RNA moleculeencodes one or more reprogramming factors. In another embodiment, theone or more reprogramming factors includes Oct4 protein. In anotherembodiment, the cell is also contacted with a synthetic RNA moleculethat encodes Sox2 protein. In yet another embodiment, the cell is alsocontacted with a synthetic RNA molecule that encodes Klf4 protein. Inyet another embodiment, the cell is also contacted with a synthetic RNAmolecule that encodes c-Myc protein. In yet another embodiment, the cellis also contacted with a synthetic RNA molecule that encodes Lin28protein.

Enzymes such as T7 RNA polymerase may preferentially incorporatecanonical nucleotides in an in vitro-transcription reaction containingboth canonical and non-canonical nucleotides. As a result, an invitro-transcription reaction containing a certain fraction of anon-canonical nucleotide may yield RNA containing a different, oftenlower, fraction of the non-canonical nucleotide than the fraction atwhich the non-canonical nucleotide was present in the reaction. Incertain embodiments, references to nucleotide incorporation fractions(for example, “a synthetic RNA molecule containing 50%pseudoisocytidine” or “0.1-0.8 pseudoisocytidine”) therefore can referboth to RNA molecules containing the stated fraction of the nucleotide,and to RNA molecules synthesized in a reaction containing the statedfraction of the nucleotide (or nucleotide derivative, for example,nucleotide-triphosphate), even though such a reaction may yield RNAcontaining a different fraction of the nucleotide than the fraction atwhich the non-canonical nucleotide was present in the reaction.

Different nucleotide sequences can encode the same protein by utilizingalternative codons. In certain embodiments, references to nucleotideincorporation fractions therefore can refer both to RNA moleculescontaining the stated fraction of the nucleotide, and to RNA moleculesencoding the same protein as a different RNA molecule, wherein thedifferent RNA molecule contains the stated fraction of the nucleotide.

Certain embodiments are directed to a kit containing one or morematerials needed to practice the present invention. In one embodiment,the kit contains synthetic RNA molecules. In one embodiment, the kitcontains synthetic RNA molecules that encode one or more reprogrammingfactors and/or gene-editing proteins. In another embodiment, thesynthetic RNA molecules contain one or more non-canonical nucleotidesthat include one or more substitutions at the 2C and/or 4C and/or 5Cpositions in the case of a pyrimidine or the 6C and/or 7N and/or 8Cpositions in the case of a purine. In another embodiment, the kitcontains one or more of: a transfection medium, a transfection reagent,a complexation medium, and a coating solution. In one embodiment, thecoating solution contains fibronectin and/or vitronectin, preferablyrecombinant fibronectin and/or recombinant vitronectin. In oneembodiment, one or more of the components of the kit are present as aplurality of aliquots. In one embodiment, the kit contains aliquots ofnucleic acid transfection-reagent complexes. In another embodiment, thekit contains aliquots of nucleic acid transfection-reagent complexesthat are provided in a solid form, for example, as frozen orfreeze-dried pellets. In yet another embodiment, the kit containsaliquots of medium, wherein each aliquot contains transfectionreagent-nucleic acid complexes that are stabilized either by chemicaltreatment or by freezing.

Transfection, in general, and reprogramming, in particular, can bedifficult and time-consuming techniques that can be repetitive and proneto error. However, these techniques are often performed manually due tothe lack of automated transfection equipment. Certain embodiments aretherefore directed to a system that can transfect, reprogram, and/orgene-edit cells in an automated or semi-automated manner.

Referring now to FIG. 9A through FIG. 11 , certain embodiments aredirected to a system (1) capable of transfecting cells in a multi-wellplate (2). In one embodiment, the plate is loaded into a tray (3) thatslides out from the system. In another embodiment, the system is capableof storing multiple plates (12). In yet another embodiment, the systemcomprises a means (4) to store a transfection medium. In one embodiment,the system comprises a means to store the medium at a definedtemperature, preferably between 2C and 6C. In one embodiment, the systemcomprises a means (5) to store liquid, waste, and/or cells removed fromthe wells. In another embodiment, the system comprises a connection tosupply power (6). In yet another embodiment, the system comprises a port(33) to communicate with a computer (34). In one embodiment, the port isa USB port. In one embodiment, the system comprises an outtake fan (7).In another embodiment, the system comprises a connection to supply avacuum (8).

Cell viability can benefit from controlling the environment around thecells. Certain embodiments are therefore directed to a system comprisinga means for incubating cells at a specified or desired temperature. Inone embodiment, the cells are incubated at one or more temperatures thatare between 35C and 39C. In one embodiment, the cells are incubated at atemperature of about 37C. Other embodiments are directed to a systemcomprising a means for controlling the atmosphere in which cells areincubated. In one embodiment, the system comprises a means forregulating the carbon dioxide concentration of the atmosphere. In oneembodiment, the carbon dioxide concentration is between 3% and 7%,preferably about 5%. In another embodiment, the system comprises a meansfor regulating the oxygen concentration of the atmosphere. In oneembodiment, the system regulates the oxygen concentration by introducingnitrogen. In still another embodiment, the oxygen concentration isbetween about 3% and about 7%, such as about 5%. In one embodiment, thesystem comprises a means for controlling both the oxygen and carbondioxide concentrations of the atmosphere in which the cells areincubated. In another embodiment, the system comprises a connection tosupply carbon dioxide (9). In yet another embodiment, the systemcomprises a connection to supply nitrogen (10). In yet anotherembodiment, the system comprises a connection to supply oxygen (11).

Certain embodiments are directed to a system comprising a means fordispensing nucleic acid transfection-reagent complexes and/or media(24). In one embodiment, the system comprises one or more front-loadedpipettes that can dispense complexes and/or media. Examples of othermeans for dispensing complexes include, but are not limited to: aback-loaded pipette, a peristaltic pump, a microfluidic device, anelectrospray nozzle, a piezoelectric ejector, and an acoustic dropletejector. Certain embodiments are directed to a system comprising a meansfor generating nucleic acid transfection-reagent complexes (13). In oneembodiment, the system comprises a means for combining one or moretransfection reagents (14) and one or more nucleic acids (15). In oneembodiment, the means for combining comprises one or more front-loadedpipettes. Examples of other means that can be used for combininginclude, but are not limited to: a back-loaded pipette, a peristalticpump, a microfluidic device, an electrospray nozzle, a piezoelectricejector, and an acoustic droplet ejector. In one embodiment, the systemcomprises one or more removable tips. In another embodiment, the one ormore removable tips can be sterilized. In another embodiment the one ormore removable tips are disposable. In yet another embodiment, the oneor more removable tips are made of plastic or glass. In still anotherembodiment, the plastic is polypropylene. In one embodiment, the systemcomprises a means for incubating one or more nucleic acids with one ormore transfection reagents in one or more complexation media (16). Inanother embodiment, the system comprises a means for storing one or morenucleic acids, one or more transfection reagents, and one or morecomplexation media. In one embodiment, the complexation occurs at roomtemperature. In one embodiment, the system comprises a means for warmingthe medium prior to contacting the cells with the medium, for example tobetween about 20° C. and about 39° C., or to between about 30° C. andabout 39° C. In one embodiment, the medium is warmed using a heatingelement (25). In one embodiment, the system comprises a means forstoring and/or dispensing multiple culture media.

Certain embodiments are directed to a method for storing nucleic acidtransfection-reagent complexes. In one embodiment, one or more nucleicacids and one or more transfection reagents are combined with one ormore complexation media and are cooled to generate a nucleic acidtransfection-reagent pellet. In one embodiment, the cooling is performedby contacting with liquid nitrogen. Other cooling methods include, butare not limited to, contacting with: a Peltier cooler, cooled liquidpropane, cooled liquid ethane, and a cooled polished metal surface. Inone embodiment, the method is substantially free of RNase. Certainembodiments are directed to a method for transfecting cells using anucleic acid transfection-reagent pellet. In one embodiment, the pelletis warmed prior to being added to the transfection medium. In oneembodiment, the pellet is warmed by placing the pellet in a small volumeof warm transfection medium that is then contacted with the cells to betransfected. In another embodiment, the pellet is added directly to thetransfection medium. Certain embodiments are directed to a system thatcan perform transfection using nucleic acid transfection-reagentpellets. In one embodiment, the system comprises a means for storing thepellets (17) within a defined temperature range. In one embodiment, thetemperature range is between about −90° C. and about 0° C., preferablybetween about −30° C. and about −4° C. In one embodiment, the systemcomprises a means for dispensing pellets. In one embodiment, the pelletsare dispensed using a plunger (19). In another embodiment, the pelletsare dispensed using a rotating disk (20) that contains an opening (21)through which the pellets are dispensed. In one embodiment, theapparatus comprises a means for warming the pellet prior to adding thepellet to the transfection medium. In one embodiment, the pellet iswarmed by placing the pellet in a small container (22) containing warmtransfection medium that is then contacted with the cells to betransfected. In another embodiment, the apparatus contains a means fordispensing the pellet directly into the transfection medium. In yetanother embodiment, the pellets are stored in a cartridge (16). In oneembodiment, the system comprises a means for replacing cartridges (36).

During cell culture it may be beneficial to replace, either in whole orin part, the culture medium or to supplement the culture medium with anadditional amount of medium or other supplement in order to addnutrients and/or to reduce, remove, or otherwise inactivate cellularwaste or other undesirable components that may be present in the medium,including residual complexes. Certain embodiments are therefore directedto a system comprising a means (23) for removing, in whole or in part,the culture medium from the cells. In one embodiment, the systemcomprises an aspirator.

Certain embodiments are directed to a system comprising a means forremoving the lid of a well plate. In one embodiment, the systemcomprises a means for removing the lid of a well plate (26) usingsuction (27). Other means for removing the lid of a well plate include,but are not limited to: an adhesive, an articulated appendage (28), aclamp, a magnet, and an electromagnet. In certain embodiments, thesystem comprises a means for imaging the cells (29). In one embodiment,the cell density is determined by measuring the optical density of thevessel containing the cells. In another embodiment, the cell density isdetermined by imaging the cells.

Certain embodiments are directed to a system that is used in operablecombination with other equipment, for example, equipment for culturing,imaging, or otherwise manipulating cells. In one embodiment, the system(1) is loaded using a robotic arm (30). In another embodiment, a roboticarm is used to transfer plates to and/or from an incubator (31). In yetanother embodiment, a plate imager (32) is used to image the cells. Inyet another embodiment, the system is controlled using a computer (34).In one embodiment, the system is used for transfecting, reprogramming,and/or gene-editing cells.

The present invention therefore has the aim of providing products forboth research and therapeutic use.

The details of the invention are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

EXAMPLES Example 1 RNA Synthesis

RNA encoding the human proteins Oct4, Sox2, Klf4, c-Myc-2 (T58A), andLin28 and comprising various combinations of canonical and non-canonicalnucleotides, was synthesized from DNA templates (Table 1). Samples ofthe RNA were analyzed by agarose gel electrophoresis to assess thequality of the RNA (FIG. 1 ). The RNA was then diluted to between 100ng/μL and 500 ng/μL. For certain experiments, an RNase inhibitor(Superase·In™, Life Technologies Corporation) was added at aconcentration of 1 μL/100 μg of RNA. RNA solutions were stored at 4C.For certain experiments involving RNA mixtures, RNA encoding Oct4, Sox2,Klf4, c-Myc-2 (T58A), and Lin28 was mixed at a molar ratio of 3:1:1:1:1.

TABLE 1 Reaction ivT Template Nucleotides Volume/μL Yield/μg Oct4 A, G,psU, 5mC 210 1976.0 Sox2 A, G, psU, 5mC 70 841.7 Klf4 A, G, psU, 5mC 70950.0 c-Myc-2 (T58A) A, G, psU, 5mC 70 535.8 Lin28 A, G, psU, 5mC 70551.0 Oct4 A, G, psU, 5mC 105 1181.8 Sox2 A, G, psU, 5mC 35 533.9 Klf4A, G, psU, 5mC 35 552.9 c-Myc-2 (T58A) A, G, psU, 5mC 35 471.2 Lin28 A,G, psU, 5mC 35 440.8 Oct4 A, G, psU, 5mC 105 1155.2 Sox2 A, G, psU, 5mC35 526.3 Klf4 A, G, psU, 5mC 35 494.0 c-Myc-2 (T58A) A, G, psU, 5mC 35446.5 Lin28 A, G, psU, 5mC 35 389.5 Sox2 A, G, psU, 5mC 20 143.8 Sox2 A,G, U, psisoC 20 114.1 Sox2 A, G, 0.25 2sU, psisoC 20 78.0 Sox2 A, G,0.25 2sU, 0.25 psisoC 20 140.1 Sox2 A, G, 5mU, psisoC 20 30.6 Sox2 A, G,0.5 5mU, psisoC 20 65.9 Oct4 A, G, U, psisoC 30 191.6 Sox2 A, G, U,psisoC 10 50.7 Klf4 A, G, U, psisoC 10 74.5 c-Myc-2 (T58A) A, G, U,psisoC 10 87.2 Lin28 A, G, U, psisoC 10 86.8 Oct4 A, G, 0.25 5mU, psisoC30 195.8 Sox2 A, G, 0.25 5mU, psisoC 10 36.2 Klf4 A, G, 0.25 5mU, psisoC10 33.6 c-Myc-2 (T58A) A, G, 0.25 5mU, psisoC 10 63.0 Lin28 A, G, 0.255mU, psisoC 10 77.2 Oct4 A, G, U, C 30 165.2 Sox2 A, G, U, C 10 94.7Klf4 A, G, U, C 10 91.4 c-Myc-2 (T58A) A, G, U, C 10 84.9 Lin28 A, G, U,C 10 104.4 Oct4 A, G, U, 0.25 psisoC 30 161.2 Sox2 A, G, U, 0.25 psisoC10 83.8 Klf4 A, G, U, 0.25 psisoC 10 85.1 c-Myc-2 (T58A) A, G, U, 0.25psisoC 10 89.3 Lin28 A, G, U, 0.25 psisoC 10 94.9 Oct4 A, G, U, 0.5psisoC 30 150.8 Sox2 A, G, U, 0.5 psisoC 10 79.3 Klf4 A, G, U, 0.5psisoC 10 83.8 c-Myc-2 (T58A) A, G, U, 0.5 psisoC 10 94.7 Lin28 A, G, U,0.5 psisoC 10 78.6 Oct4 0.25 7dA, G, U, C 10 29.7 Oct4 0.5 7dA, G, U, C10 44.7 Oct4 A, 0.25 7dG, U, C 10 45.2 Oct4 A, 0.5 7dG, U, C 10 31.7Oct4 0.25 7dA, 0.25 7dG, U, C 10 13.2 Oct4 0.25 7dA, G, U, 0.25 psisoC10 47.6 Oct4 A, 0.25 7dG, U, 0.25 psisoC 10 10.5 Oct4 A, 0.5 7dG, U,0.25 psisoC 30 125.3 Sox2 A, 0.5 7dG, U, 0.25 psisoC 10 20.5 Klf4 A, 0.57dG, U, 0.25 psisoC 10 18.4 c-Myc-2 (T58A) A, 0.5 7dG, U, 0.25 psisoC 1022.1 Lin28 A, 0.5 7dG, U, 0.25 psisoC 10 39.7 Oct4 A, 0.5 7dG, U, 0.5psisoC 30 92.3 Sox2 A, 0.5 7dG, U, 0.5 psisoC 10 20.1 Klf4 A, 0.5 7dG,U, 0.5 psisoC 10 17.7 c-Myc-2 (T58A) A, 0.5 7dG, U, 0.5 psisoC 10 95.4Lin28 A, 0.5 7dG, U, 0.5 psisoC 10 26.0 Oct4 0.25 7dA, 7dG, U, 0.25psisoC 20 3.8 Sox2 0.25 7dA, 7dG, U, 0.25 psisoC 20 5.4 Klf4 0.25 7dA,7dG, U, 0.25 psisoC 20 5.9 c-Myc-2 (T58A) 0.25 7dA, 7dG, U, 0.25 psisoC20 5.9 Lin28 0.25 7dA, 7dG, U, 0.25 psisoC 20 5.1 Oct4 0.25 7dA, 7dG, U,0.5 psisoC 20 3.0 Sox2 0.25 7dA, 7dG, U, 0.5 psisoC 20 3.3 Klf4 0.257dA, 7dG, U, 0.5 psisoC 20 4.1 c-Myc-2 (T58A) 0.25 7dA, 7dG, U, 0.5psisoC 20 4.5 Lin28 0.25 7dA, 7dG, U, 0.5 psisoC 20 5.0 Oct4 A, 0.757dG, U, C 10 40.8 (2 h incubation) Oct4 A, 7dG, U, C 10 14.1 (2 hincubation) Oct4 A, 0.75 7dG, U, C 10 42.9 (20 h incubation) Oct4 A,7dG, U, C 10 24.4 (20 h incubation) Oct4 A, G, U, 0.25 N4mC 10 73.1 Oct4A, G, U, 0.5 N4mC 10 66.2 Oct4 A, G, U, 0.75 N4mC 10 55.1 Oct4 A, G, U,N4mC 10 32.7 Oct4 A, 0.75 7dG, U, C 10 35.6

“A” refers to adenosine-5′-triphosphate, “G” refers toguanosine-5′-triphosphate, “U” refers to uridine-5′-triphosphate, “C”refers to cytidine-5′-triphosphate, “psU” refers topseudouridine-5′-triphosphate, “5mC” refers to5-methylcytidine-5′-triphosphate, “2sU” refers to2-thiouridine-5′-triphosphate, “psisoC” refers topseudoisocytidine-5′-triphosphate, “5mU” refers to5-methyluridine-5′-triphosphate, “7dA” refers to7-deazaadenosine-5′-triphosphate, “7dG” refers to7-deazaguanosine-5′-triphosphate, and “N4mC” refers toN4-methylcytidine-5′-triphosphate.

Example 2 Transfection Medium Formulation

A medium was developed to support efficient transfection, reprogramming,and gene-editing of cells:

DMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodiumselenite+20 ng/mL bFGF+5 mg/mL treated human serum albumin.

Variants of this medium were also developed to provide improvedperformance when used with specific transfection reagents, specificnucleic acids, and specific cell types: DMEM/F12+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+4.5 μg/mL cholesterol+20ng/mL bFGF+5 mg/mL treated human serum albumin, DMEM/F12+10 μg/mLinsulin+5.5 μg/mL transferrin+6.7 ng/mL sodium selenite+1 μMhydrocortisone+20 ng/mL bFGF+5 mg/mL treated human serum albumin, andDMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodiumselenite+4.5 μg/mL cholesterol+1 μM hydrocortisone+20 ng/mL bFGF+5 mg/mLtreated human serum albumin.

Examples of additional components that were added to the cell-culturemedium in certain experiments (listed with example concentrations)include: 15 mM HEPES, 2 mM L-alanyl-L-glutamine, 2 μg/mL ethanolamine,10 μg/mL fatty acids, 10 μg/mL cod liver oil fatty acids (methylesters), 25 μg/mL polyoxyethylenesorbitan monooleate, 2 μg/mLD-alpha-tocopherol acetate, 1-50 μg/mL L-ascorbic acid 2-phosphatesesquimagnesium salt hydrate, 200 ng/mL B18R, and 0.1% Pluronic F-68.

For certain experiments in which the medium was conditioned, thefollowing variant was used:

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+10 μg/mL cod liver oil fatty acids (methyl esters)+25μg/mL polyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherolacetate+1 μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salthydrate+0.1% Pluronic F-68+20 ng/mL bFGF+5 mg/mL treated human serumalbumin.

For certain experiments in which the medium was not conditioned, thefollowing variant was used.

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+4.5μg/mL cholesterol+1 μM hydrocortisone+0-25 μg/mL polyoxyethylenesorbitanmonooleate+2 μg/mL D-alpha-tocopherol acetate+50 μg/mL L-ascorbic acid2-phosphate sesquimagnesium salt hydrate+20 ng/mL bFGF+5 mg/mL treatedhuman serum albumin.

For the preparation of the these variants, the treated human serumalbumin was treated by addition of 32 mM sodium octanoate, followed byheating at 60C for 4 h, followed by treatment with ion-exchange resin(AG501-X8(D)) for 6 h at room temperature, followed by treatment withdextran-coated activated charcoal (C6241, Sigma-Aldrich Co. LLC.)overnight at room temperature, followed by centrifugation, filtering,adjustment to a 10% solution with nuclease-free water, followed byaddition to the other components of the medium. For certain experimentsin which the medium was conditioned, the medium was conditioned for 24 hon irradiated human neonatal fibroblast feeders. The cells were platedon fibronectin-coated plates or fibronectin and vitronectin-coatedplates, unless otherwise noted.

The formulation of the medium can be adjusted to meet the needs of thespecific cell types being cultured. Furthermore, in certain situations,treated human serum albumin can be replaced with other treated albumin,for example, treated bovine serum albumin, other glutamine sources canbe used instead of or in addition to L-alanyl-L-glutamine, for example,L-glutamine, other buffering systems can be used instead of or inaddition to HEPES, for example, phosphate, bicarbonate, etc., seleniumcan be provided in other forms instead of or in addition to sodiumselenite, for example, selenous acid, other antioxidants can be usedinstead of or in addition to L-ascorbic acid 2-phosphate sesquimagnesiumsalt hydrate and/or D-alpha-tocopherol acetate, for example, L-ascorbicacid, other surfactants can be used instead of or in addition topolyoxyethylenesorbitan monooleate and/or Pluronic F-68, for example,Pluronic F-127, other basal media can be used instead of or in additionto DMEM/F12, for example, MEM, DMEM, etc., and the components of theculture medium can be varied with time, for example, by using a mediumwithout TGF-β from day 0 to day 5, and then using a medium containing 2ng/mL TGF-β after day 5. In certain situations, other ingredients can beadded, for example, fatty acids, lysophosphatidic acid,lysosphingomyelin, sphingosine-1-phosphate, other sphingolipids, membersof the TGF-β/NODAL family of proteins, IL-6, members of the Wnt familyof proteins, etc., at appropriate concentrations, and ingredients thatare known to promote or inhibit the growth of specific cell types and/oragonists and/or antagonists of proteins or other molecules that areknown to promote or inhibit the growth of specific cell types can beadded to the medium at appropriate concentrations when it is used withthose cell types, for example, sphingosine-1-phosphate and pluripotentstem cells. Ingredients can take the form of purified compounds, partsof well-defined mixtures, parts of complex or undefined mixtures, forexample, animal or plant oils, and may be added by biological processes,for example, conditioning. The concentrations of the components can bevaried from the listed values within ranges that will be obvious topersons skilled in the art.

Example 3 Transfection of Cells with Synthetic RNA

For transfection in 6-well plates, 2 μg RNA and 6 μL transfectionreagent (Lipofectamine™ RNAiMAX, Life Technologies Corporation) werefirst diluted separately in complexation medium (Opti-MEM®, LifeTechnologies Corporation) to a total volume of 60 μL each. Diluted RNAand transfection reagent were then mixed and incubated for 15 min atroom temperature, according to the transfection reagent-manufacturer'sinstructions. Complexes were then added to cells in culture. Between 30μL and 240 μL of complexes were added to each well of a 6-well plate,which already contained 2 mL of transfection medium per well. Plateswere then shaken gently to distribute the complexes throughout the well.Cells were incubated with complexes for 2 hours to overnight, beforereplacing the medium with fresh transfection medium (2 mL/well). Volumeswere scaled for transfection in 24-well and 96-well plates. Cells werefixed and stained 20-24 h after transfection using an antibody againstOct4 (FIG. 2A). Nuclei were stained and counted to determine therelative toxicity of the RNA (FIG. 2B).

Example 4 Analysis of the Ability of Untreated Human Serum AlbuminPreparations to Support Nucleic Acid Transfection and RNA Reprogramming

Primary human neonatal fibroblasts were cultured in medium with orwithout 5 mg/mL HSA. Cohn Fraction V (A6784, Sigma-Aldrich Co. LLC.),and four different recombinant HSA preparations (A6608, A7736, A9731,and A9986, all from Sigma-Aldrich Co. LLC.) were screened. Cells weretransfected according to Example 3, with RNA synthesized according toExample 1. While untransfected cells grew well in media containing anyof the HSA preparations, in transfected wells, each of the HSApreparations yielded dramatically different cell morphologies and celldensities, and none resulted in morphological changes indicative ofreprogramming.

Example 5 Production of Octanoate-Treated Human Serum Albumin

A 10% solution of HSA was pre-incubated with 22 mM sodium chloride and16 mM sodium octanoate (Sigma-Aldrich Co. LLC), and was incubated at 37Cfor 3 hours before assembly of the complete medium.

Example 6 Treatment of Human Serum Albumin Using Ion-ExchangeChromatography

A 20% solution of recombinant HSA produced in Pichia pastoris (A7736,Sigma-Aldrich Co. LLC.) was prepared by dissolving 2 g of HSA in 10 mLof nuclease-free water with gentle agitation at room temperature. TheHSA solution was then deionized by first adding 1 g of mixed-beddeionizing resin (AG 501-X8(D), Bio-Rad Laboratories, Inc.), and rockingfor 1 h at room temperature. The HSA solution was then decanted into atube containing 5 g of fresh resin, and was rocked for 4 h at roomtemperature. Finally, the deionized HSA solution was decanted, adjustedto a 10% total protein content with nuclease-free water,filter-sterilized using a 0.2 μm PES-membrane filter, and stored at 4C.

Example 7 Analysis of Transfection Efficiency and Viability of CellsCultured in Media Containing Octanoate-Treated Human Serum Albumin

Primary human neonatal fibroblasts were cultured in media containingrecombinant HSA treated according to Example 4 or containing treatedblood-derived HSA (Bio-Pure HSA, Biological Industries). Cells weretransfected daily, according to Example 3, with RNA synthesizedaccording to Example 1, beginning on day 0. Pictures were taken on day3. Several small areas of cells undergoing morphological changesresembling mesenchymal to epithelial transition were observed in thewells containing octanoate, indicating an increased transfectionefficiency. Many large areas of morphological changes resemblingmesenchymal to epithelial transition were observed in the samplescontaining the treated blood-derived HSA. In both cases, themorphological changes were characteristic of reprogramming

Example 8 Reprogramming Human Fibroblasts Using Media ContainingOctanoate-Treated Human Serum Albumin

Primary human neonatal fibroblasts were plated in 6-well plates at adensity of 5000 cells/well in fibroblast medium (DMEM+10% fetal bovineserum). After 6 hours, the medium was replaced with transfection mediumcontaining octanoate-treated HSA. The cells were transfected daily,according to Example 3, with RNA synthesized according to Example 1,beginning on day 0. By day 5, the well contained several areas of cellsexhibiting morphology consistent with reprogramming. This experiment didnot include the use of feeders or immunosuppressants.

Example 9 Analysis of Transfection Efficiency and Viability of CellsCultured in Media Containing Ion-Exchange-Resin-Treated Human SerumAlbumin

Primary human neonatal fibroblasts were transfected according to Example3, with RNA synthesized according to Example 1, beginning on day 0.Pictures were taken on day 2. Cells in the well containing untreated HSAexhibited low viability compared to either the well containing treatedblood-derived HSA or ion-exchange-resin-treated recombinant HSA.

Example 10 Reprogramming Human Fibroblasts UsingIon-Exchange-Resin-Treated Human Serum Albumin

Primary human neonatal fibroblasts were plated in 6-well plates onfeeders at a density of 10,000 cells/well in fibroblast medium (DMEM+10%fetal bovine serum). The cells were transfected daily according toExample 3, with RNA synthesized according to Example 1, beginning on day0. A passage with a split ratio of 1:20 was performed on day 4. Pictureswere taken on day 10. The well contained many large colonies of cellsexhibiting morphology consistent with reprogramming. No colonies wereobserved in wells exposed to cell-culture media containing untreatedHSA.

Example 11 Reprogramming Human Fibroblasts without Using Feeders orImmunosuppressants

Primary human fibroblasts were plated in 6-well plates at a density of20,000 cells/well in fibroblast medium (DMEM+10% fetal bovine serum).After 6 hours, the medium was replaced with transfection mediumcontaining treated HSA and not containing immunosuppressants, and thecells were transfected daily according to Example 3, with RNAsynthesized according to Example 1, except that the dose of RNA wasreduced to 1 μg/well and a total of 5 transfections were performed.Pictures were taken on day 7. Small colonies of cells exhibitingmorphology consistent with reprogramming became visible as early as day5. On day 7, the medium was replaced with DMEM/F12+20% Knockout™ SerumReplacement (Life Technologies Corporation)+1×non-essential aminoacids+2 mM L-glutamine, conditioned on irradiated mouse embryonicfibroblasts for 24 hours, and then supplemented with 20 ng/mL bFGF and10 μM Y-27632. Large colonies with a reprogrammed morphology becamevisible as early as day 8. Colonies were picked on day 10, and plated inwells coated with basement membrane extract (Cultrex® Human BMEPathclear®, Trevigen Inc.) (FIG. 3A). Cells grew rapidly, and werepassaged to establish lines. Established lines stained positive for thepluripotent stem-cell markers Oct4 and SSEA4 (FIG. 3B). The entireprotocol was repeated, and similar results were obtained (FIG. 3C).

Example 12 Efficient, Rapid Derivation and Reprogramming of Cells fromHuman Skin Biopsy Tissue

A full-thickness dermal punch biopsy was performed on a healthy, 31year-old volunteer, according to an approved protocol. Briefly, an areaof skin on the left, upper arm was anesthetized by topical applicationof 2.5% lidocaine. The field was disinfected with 70% isopropanol, and afull-thickness dermal biopsy was performed using a 1.5 mm-diameter punch(FIG. 4A). The tissue was rinsed in phosphate-buffered saline (PBS), andwas placed in a 1.5 mL tube containing 250 μL of TrypLE™ Select CTS™(Life Technologies Corporation), and incubated at 37C for 30 min. Thetissue was then transferred to a 1.5 mL tube containing 250 μL ofDMEM/F12-CTS™ (Life Technologies Corporation)+5 mg/mL collagenase, andincubated at 37C for 2 h (FIG. 4B). The epidermis was removed usingforceps, and the tissue was mechanically dissociated. Cells were rinsedtwice in DMEM/F12-CTS™ and were plated in fibronectin-coated wells of24-well and 96-well plates. Phlebotomy was also performed on the samevolunteer, and venous blood was collected in Vacutainer® SST™ tubes(Becton, Dickinson and Company). Serum was isolated according to themanufacturer's protocol. Isogenic plating medium was prepared by mixingDMEM/F12-CTS™+2 mM L-alanyl-L-glutamine (Sigma-Aldrich Co. LLC.)+20%human serum. Cells from the dermal tissue sample were plated either intransfection medium or in isogenic plating medium. After 2 days, thewells were rinsed, and the medium was replaced with transfection medium.Many cells with a fibroblast morphology attached and began to spread byday 2 (FIG. 4C). Cells were transfected according to Example 3, with RNAsynthesized according to Example 1, beginning on day 2, with all volumesscaled to accommodate the smaller wells. By day 5, areas of cells withmorphologies consistent with reprogramming were observed.

Example 13 Reprogramming Human Fibroblasts Using Synthetic RNAContaining Non-Canonical Nucleotides

Primary human fibroblasts were plated in 6-well plates coated withrecombinant human fibronectin and recombinant human vitronectin (eachdiluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, incubatedat room temperature for 1 h) at a density of 20,000 cells/well intransfection medium. The following day, the cells were transfected as inExample 3, with RNA synthesized according to Example 1, except that thedose of RNA was 0.5 μg/well on day 1, 0.5 μg/well on day 2, and 2μg/well on day 3. Pictures were taken on day 4. Small colonies of cellsexhibiting morphology consistent with reprogramming were visible on day4.

Example 14 Reprogramming Human Fibroblasts with a Non-ConditionedTransfection Medium

Primary human fibroblasts were plated in 6-well plates coated withrecombinant human fibronectin and recombinant human vitronectin (eachdiluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, incubatedat room temperature for 1 h) at a density of 20,000 cells/well intransfection medium. The following day, the cells were transfected as inExample 3, with RNA synthesized according to Example 1, except that thedose of RNA was 0.5 μg/well on day 1, 0.5 μg/well on day 2, 2 g/well onday 3, 2 μg/well on day 4, and 4 μg/well on day 5. Small colonies ofcells exhibiting morphology consistent with reprogramming became visibleas early as day 5. On day 7, the medium was replaced with DMEM/F12+20%Knockout™ Serum Replacement (Life Technologies Corporation)+1×non-essential amino acids+2 mM L-glutamine, conditioned on irradiatedmouse embryonic fibroblasts for 24 hours, and then supplemented with 20ng/mL bFGF and 10 μM Y-27632. Large colonies with a reprogrammedmorphology became visible as early as day 8. Colonies were picked on day10, and plated in wells coated with basement membrane extract (Cultrex®Human BME Pathclear®, Trevigen Inc.). Cells grew rapidly, and werepassaged to establish lines.

Example 15 Generation of Glucose-Responsive Insulin-Producing Cells

Cells are reprogrammed according to Example 11 or Example 12, and arethen cultured in DMEM/F12+0.2% HSA+0.5× N2 supplement+0.5× B27supplement+100 ng/mL activin A+1 μM wortmannin for 4 days, followed by1:1 F12/IMDM+0.5% HSA+0.5% ITS supplement+0.5× B27 supplement+2 μMretinoic acid+20 ng/mL FGF7+50 ng/mL NOGGIN for 4 days, followed byDMEM+0.5% HSA+1% ITS supplement+1× N2 supplement+50 ng/mL EGF for 5days, followed by DMEM/F12+1% ITS supplement+10 ng/mL bFGF+10 mMnicotinamide+50 ng/mL exendin-4+10 ng/mL BMP4 for 7-9 days to generateglucose-responsive insulin-producing cells. Alternatively, cells arereprogrammed according to Example 11 or Example 12, and are thencultured in 1:1 F12/IMDM+0.5% HSA+0.5% ITS supplement+0.5× B27supplement+2 μM retinoic acid+20 ng/mL FGF7+50 ng/mL NOGGIN for 4 days,followed by DMEM+0.5% HSA+1% ITS supplement+1× N2 supplement+50 ng/mLEGF for 5 days, followed by DMEM/F12+1% ITS supplement+10 ng/mL bFGF+10mM nicotinamide+50 ng/mL exendin-4+10 ng/mL BMP4 for 7-9 days togenerate glucose-responsive insulin-producing cells, without generatingdefinitive endoderm cells. Alternatively, cells are reprogrammedaccording to Example 11 or Example 12, and are then cultured in 1:1F12/IMDM+0.5% HSA+0.5% ITS supplement+0.5× B27 supplement+2 μM retinoicacid+20 ng/mL FGF7+50 ng/mL NOGGIN for 4 days, followed by DMEM/F12+1%ITS supplement+10 ng/mL bFGF+10 mM nicotinamide+50 ng/mL exendin-4+10ng/mL BMP4 for 7-9 days to generate glucose-responsive insulin-producingcells, without generating definitive endoderm cells, and withoutexpanding progenitor cells. While endodermal cells or insulin-producingcells can be isolated from other cells present in the culture, thismethod generates a sufficiently high percentage of glucose-responsiveinsulin producing cells that such isolation is not generally required.The resulting cells can then be used in vitro or in vivo for screeningbioactive molecules for the study of diabetes or for the development oftherapeutics for diabetes.

Example 16 Generation of Glucose-Responsive Insulin-Producing CellsUsing Recombinant Proteins

Cells were reprogrammed according to Example 11, and were then culturedin DMEM/F12, 100 ng/ml activin A, 25 ng/ml Wnt3a, 0.01% recombinant HSA,1× ITSE for 1 day, followed by DMEM/F12, 100 ng/ml activin A, 0.01%recombinant HSA, 1× ITSE for 2 days, followed by DMEM/F12, 50 ng/mlFGF10, 0.25 μM KAAD-cyclopamine, 0.01% recombinant HSA, 1× ITSE for 3days, followed by DMEM/F12, 1% B27, 2 μM all-trans retinoic acid, 50ng/ml FGF10, 0.25 μM KAAD-cyclopamine for 4 days, followed by DMEM/F12,1% B27, 1 μM γ-secretase inhibitor DAPT, 50 ng/ml exendin-4, 10 nMbetacellulin, 10 mM nicotinamide for 2 days, followed by DMEM/F12, 50mg/L ascorbic-acid-2-phosphate, 1% B27, 1 μM γ-secretase inhibitor DAPT,50 ng/ml exendin-4, 50 ng/ml IGF-1, 50 ng/ml HGF, 10 nM betacellulin, 10mM nicotinamide for 6 days to generate glucose-responsiveinsulin-producing cells (FIG. 5A). The resulting cells can be used invitro or in vivo for screening bioactive molecules for the study ofdiabetes or for the development of therapeutics for diabetes.

Example 17 Personalized Cell-Replacement Therapy for Type 1 DiabetesComprising Reprogrammed Cells

Patient skin cells are reprogrammed to glucose-responsiveinsulin-producing cells according to Example 12 and Example 14. Cellsare then enzymatically released from the culture vessel, and betweenabout 1×10⁶ and about 1×10⁷ cells are injected into the intraperitonealspace or into the portal vein. In the case of intraperitoneal injection,cells are pre-mixed with an extracellular matrix protein to preventexcessive migration. Cells engraft and begin producing insulin.Insulin/C-peptide levels are monitored, and additional injections areperformed as necessary.

Example 18 Synthesis of RNA TALENs

RNA encoding 20 bp-matching TALENs was synthesized from DNA templates asin Example 1 (FIG. 6A-C and FIG. 7 ) (Table 2). The resulting RNA wasanalyzed by agarose gel electrophoresis to assess the quality of theRNA. The RNA was then diluted to 200 ng/μL, and an RNase inhibitor(Superase·In™, Life Technologies Corporation) was added at aconcentration of 1 μL/100 μg of RNA. RNA solutions were stored at 4C.RNA encoding each half of the TALEN pair was mixed at a molar ratio of1:1.

TABLE 2 Reaction ivT Template Nucleotides Volume/μL Yield/μg XPA-L1 A,G, psU, 5mC 20 120.0 XPA-L2 A, G, psU, 5mC 20 114.0 XPA-R1 A, G, psU,5mC 20 159.6 CCR5-L1 A, G, psU, 5mC 20 170.4 CCR5-L2 A, G, psU, 5mC 20142.8 CCR5-R1 A, G, psU, 5mC 20 132.0 CCR5-R2 A, G, psU, 5mC 20 154.8CCR5-L1 A, G, psU, 5mC 10 56.6 CCR5-L2 A, G, psU, 5mC 10 58.5 CCR5-R1 A,G, psU, 5mC 10 56.8 CCR5-R2 A, G, psU, 5mC 10 58.7

Example 19 Synthesis of RNA TALENs Targeting the CCR5 Gene

RNA encoding the TALENs L1: TCATTTTCCATACAGTCAGT, L2:TTTTCCATACAGTCAGTATC, R1: TGACTATCTTTAATGTCTGG, and R2:TATCTTTAATGTCTGGAAAT was synthesized according to Example 18. TheseTALENs target 20-bp sites within the CCR5 gene on the sense (L1 and L2)or antisense strand (R1 and R2). The following TALEN pairs wereprepared: L1&R1, L1&R2, L2&R1, and L2&R2.

Example 20 Gene-Editing of the CCR5 Gene Using RNA TALENs and DNA-Free,Feeder-Free, Immunosuppressant-Free, Conditioning-Free Reprogramming ofHuman Fibroblasts

Primary human fibroblasts were plated in 6-well plates coated withrecombinant human fibronectin and recombinant human vitronectin (eachdiluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, incubatedat room temperature for 1 h) at a density of 10,000 cells/well intransfection medium. The following day, the cells were transfected as inExample 3, except that the dose of RNA was 0.5 μg/well, and the RNA wassynthesized according to Example 19. Beginning the following day, thecells were reprogrammed according to Example 11. Large colonies of cellswith a morphology characteristic of reprogramming became visible as inExample 11. Pictures were taken on day 9 (FIG. 8 ).

Example 21 Transfection of Cells with RNA TALENs and a DNA RepairTemplate

0.5 ug RNA+0.5 ug DNA containing the 1001 bp-region spanning from 500 bpupstream of the targeted double-strand break location to 500 bpdownstream of the targeted double-strand break location and 6 μLtransfection reagent (Lipofectamine™ 2000, Life TechnologiesCorporation) are first diluted separately in complexation medium(Opti-MEM®) to a total volume of 60 μL each. Diluted RNA+DNA andtransfection reagent are then mixed and incubated for 15 min at roomtemperature, according to the transfection reagent-manufacturer'sinstructions. Complexes are then added to cells in culture. Between 60μL and 120 μL are added to each well of a 6-well plate, which alreadycontains 2 mL of transfection medium per well. Plates are then shakengently to distribute the complexes throughout the well. Cells areincubated with complexes for 2 hours to overnight, before replacing themedium with fresh transfection medium (2 mL/well).

Example 22 Gene Editing Using RNA TALENs and a DNA Repair Template andDNA-Free, Feeder-Free, Immunosuppressant-Free, Conditioning-FreeReprogramming of Human Fibroblasts

Primary human fibroblasts are plated in 6-well plates at a density of10,000 cells/well in fibroblast medium (DMEM+10% fetal bovine serum).After 6 hours, the medium is replaced with transfection mediumcontaining treated HSA and not containing immunosuppressants, and thecells are transfected according to Example 21. Beginning the followingday, the cells are reprogrammed according to Example 11 or Example 12,except that the initial plating and media change steps are omitted.

Example 23 Generation of Hematopoietic Cells

Cells were reprogrammed according to Example 11, and were then culturedin IMDM+0.5% HSA+1× ITS supplement+450 μM monothioglycerol+2 mML-glutamine+1× non-essential amino acids+50 ng/mL BMP4+50 ng/mL VEGF+50ng/mL bFGF for 6 days to generate hematopoietic cells (FIG. 5B).Alternatively, cells are reprogrammed according to Example 11 or Example12 or Example 20 or Example 22, and are then cultured in IMDM+0.5%HSA+1× ITS supplement+450 μM monothioglycerol+2 mM L-glutamine+1×non-essential amino acids+50 ng/mL BMP4+50 ng/mL VEGF+50 ng/mL bFGF for6 days, followed by IMDM+0.5% HSA+1× ITS supplement+0.1 mM2-mercaptoethanol+5U/mL heparin+10 ng/mL TPO+25 ng/mL SCF+25 ng/mLFLT3L+10 ng/mL IL-3+10 ng/mL IL-6 for 8 days to generate hematopoieticcells. Alternatively, cells are reprogrammed according to Example 11 orExample 12 or Example 20 or Example 22, and are then re-plated oncollagen IV and cultured in IMDM+0.5% HSA+1× ITS supplement+450 μMmonothioglycerol+2 mM L-glutamine+1× non-essential amino acids+50 ng/mLBMP4+50 ng/mL VEGF+50 ng/mL bFGF for 6 days, followed by IMDM+0.5%HSA+1× ITS supplement+0.1 mM 2-mercaptoethanol+5U/mL heparin+10 ng/mLTPO+25 ng/mL SCF+25 ng/mL FLT3L+10 ng/mL IL-3+10 ng/mL IL-6 for 8 daysto generate hematopoietic cells. Alternatively, cells are reprogrammedaccording to Example 11 or Example 12 or Example 20 or Example 22, andare then cultured in 1:1 F12/IMDM+0.5% HSA+1× ITS supplement+4.5 μg/mLcholesterol+10 μg/mL cod liver oil fatty acids+25 μg/mLpolyoxyethylenesorbitan monooleate+2 μg/mL D-α-tocopherol acetate+450 μMmonothioglycerol+2 mM L-glutamine+25 ng/mL BMP4+25 ng/mL VEGF+25 ng/mLbFGF+20 ng/mL SCF for 10 days to generate hematopoietic cells.

Example 24 Personalized Cell-Replacement Therapy for Blood DiseaseComprising Reprogrammed Cells

Patient skin cells are reprogrammed to hematopoietic cells according toExample 23. Cells are then released from the culture vessel, and betweenabout 1×10⁶ and about 1×10⁷ cells/kg patient body weight are infusedinto a main vein over a period of several hours.

Example 25 Personalized Cell-Replacement Therapy for HIV/AIDS ComprisingGene-Edited and Reprogrammed Cells

Patient skin cells are gene-edited and reprogrammed to hematopoieticcells according to Example 23. Cells are then enzymatically releasedfrom the culture vessel, and between about 1×10⁶ and about 1×10⁷cells/kg patient body weight are infused into a main vein over a periodof several hours. Hematopoietic stem cells home to the bone marrowcavity and engraft. Alternatively, patient skin cells are gene-editedand reprogrammed to hematopoietic cells according to Example 23, cellsare then enzymatically released from the culture vessel, andCD34+/CD90+/Lin- or CD34+/CD49f+/Lin-cells. are isolated. Between about1×10³ and about 1×10⁵ cells are infused into a main vein of the patient.Hematopoietic stem cells home to the bone marrow cavity and engraft.

Example 26 Cardiac Disease Models for Screening Bioactive Molecules

Cells were reprogrammed according to Example 11, and were then culturedin DMEM/F12+0.2% HSA+0.5× N2 supplement+0.5× B27 supplement+100 ng/mLactivin A+1 μM wortmannin for 4 days, followed by 1:1 F12/IMDM+0.5%HSA+0.5% ITS supplement+0.5× B27 supplement+2 μM retinoic acid+20 ng/mLFGF7+50 ng/mL NOGGIN for 4 days, followed by DMEM/F12+1% ITSsupplement+10 ng/mL bFGF+10 mM nicotinamide+50 ng/mL exendin-4+10 ng/mLBMP4 for 7-9 days to generate cardiac cells (FIG. 5C). Alternatively,cells are reprogrammed according to Example 12. While cardiac cells canbe isolated from other cells present in the culture, this methodgenerates a sufficiently high percentage of cardiac cells that suchisolation is not generally required. The resulting cells can be used invitro or in vivo for screening bioactive molecules for the study ofheart disease or for the development of therapeutics for heart disease.The resulting cells can also be used for cardiotoxicity screening.

Example 27 Personalized Cell-Replacement Therapy for IschemicCardiomyopathy Comprising Reprogrammed Cells

Patient skin cells are reprogrammed to cardiac cells according toExample 26. Cells are then enzymatically released from the culturevessel, and between about 1×10⁶ and about 1×10⁷ cells are injected intothe pericardium or between about 1×10³ and about 1×10⁵ cells areinjected into one or more coronary arteries. Cells engraft, andadditional injections are performed as necessary.

Example 28 Retinal Disease Models for Screening Bioactive Molecules

Cells are reprogrammed according to Example 11 or Example 12, and arethen cultured in DMEM/F12+0.2% HSA+0.5× N2 supplement+0.5× B27supplement 7 days to generate retinal cells. The resulting cells can beused in vitro or in vivo for screening bioactive molecules for the studyof retinal disease or for the development of therapeutics for retinaldisease.

Example 29 Personalized Cell-Replacement Therapy for MacularDegeneration Comprising Reprogrammed Cells

Patient skin cells are reprogrammed to retinal cells according toExample 28. Cells are then enzymatically released from the culturevessel, and between about 1×10⁴ and about 1×10⁵ cells are injected intoor below the retina. Cells engraft, and additional injections areperformed as necessary.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

What is claimed is:
 1. A method for producing a gene-edited cell,comprising: (a) culturing a human non-pluripotent cell comprising atarget DNA sequence; and (b) transfecting the cell with a plurality ofsynthetic RNA molecules, wherein the synthetic RNA molecules include: i.a first synthetic RNA molecule encoding a first fusion proteincomprising a DNA-binding domain and a catalytic domain of a nuclease;and ii. a second synthetic RNA molecule encoding a second fusion proteincomprising a DNA-binding domain and a catalytic domain of a nuclease;wherein: the first fusion protein and the second fusion protein areindependently a transcription activator-like effector nuclease (TALEN);the transfecting results in the cell expressing the first fusion proteinand the second fusion protein to result in a single-strand break or adouble-strand break in the target DNA sequence; and the first syntheticRNA molecule and the second synthetic RNA molecule are independentlysynthesized by in vitro transcription from a DNA template.
 2. The methodof claim 1, further comprising contacting the cell with at least one ofpoly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin,collagen, and laminin.
 3. The method of claim 1, further comprisingcontacting the cell with a medium.
 4. A method for producing agene-edited cell comprising an inserted DNA sequence, comprising: (a)culturing a human non-pluripotent cell comprising a target DNA sequence;(b) transfecting the cell with a plurality of synthetic RNA molecules,wherein the synthetic RNA molecules include: i. a first synthetic RNAmolecule encoding a first fusion protein comprising a DNA-binding domainand a catalytic domain of a nuclease; and ii. a second synthetic RNAmolecule encoding a second fusion protein comprising a DNA-bindingdomain and a catalytic domain of a nuclease; wherein the first fusionprotein and the second fusion protein are independently a transcriptionactivator-like effector nuclease (TALEN), the transfecting results inthe cell expressing the first fusion protein and the second fusionprotein to result in a single-strand break or a double-strand break inthe target DNA sequence; and wherein the first synthetic RNA moleculeand the second synthetic RNA molecule are independently synthesized byin vitro transcription from a DNA template; and (c) transfecting thecell with a DNA repair template comprising a sequence for insertion andone or more regions of homology to the DNA of the cell, wherein the oneor more regions of homology comprise regions upstream and/or downstreamof the single-strand break or the double-strand break, to result ininsertion of the sequence in the region of the single-strand break orthe double-strand break.
 5. The method of claim 4, further comprisingcontacting the cell with at least one of poly-L-lysine,poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, andlaminin.
 6. The method of claim 4, further comprising contacting thecell with a medium.
 7. The method of claim 1, wherein the humannon-pluripotent cell is a skin cell or a hematopoietic cell.
 8. Themethod of claim 7, wherein the hematopoietic cell is a hematopoieticstem cell or a white blood cell.
 9. The method of claim 8, wherein theskin cell is obtained from a biopsy.
 10. The method of claim 7, whereinthe skin cell is a fibroblast, a keratinocyte, a melanocyte, anadipocyte, a mesenchymal stem cell, an adipose stem cell, or a bloodcell.
 11. The method of claim 1, wherein the plurality of synthetic RNAmolecules are complexed with a transfection reagent.
 12. The method ofclaim 11, wherein the transfection reagent is lipid-based,polymer-based, or peptide-based.
 13. The method of claim 12, wherein thetransfection reagent comprises a charged polymer or a cell-penetratingpeptide.
 14. The method of claim 12, wherein the transfection reagentcomprises a cationic lipid, a liposome, or a micelle.
 15. The method ofclaim 3, wherein the medium is substantially free of immunosuppressants.16. The method of claim 1, wherein the first synthetic RNA molecule, thesecond synthetic RNA molecule, or both the first synthetic RNA moleculeand the second synthetic RNA molecule comprise at least onenon-canonical nucleotide selected from the group consisting of: a5-methyluridine residue, a pseudouridine residue, a5-methylpseudouridine residue, a 5-hydroxyuridine residue, a5-hydroxypseudouridine residue, and a 5-methylcytidine residue.
 17. Themethod of claim 1, wherein the first synthetic RNA molecule, the secondsynthetic RNA molecule, or both the first synthetic RNA molecule and thesecond synthetic RNA molecule further comprise one or more of a 5′-cap,a 5′-cap 1 structure, and a 3′-poly(A) tail.
 18. The method of claim 4,wherein the human non-pluripotent cell is a skin cell or a hematopoieticcell.
 19. The method of claim 18, wherein the hematopoietic cell is ahematopoietic stem cell or a white blood cell.
 20. The method of claim18, wherein the skin cell is obtained from a biopsy.
 21. The method ofclaim 18, wherein the skin cell is a fibroblast, a keratinocyte, amelanocyte, an adipocyte, a mesenchymal stem cell, an adipose stem cell,or a blood cell.
 22. The method of claim 4, wherein the plurality ofsynthetic RNA molecules are complexed with a transfection reagent. 23.The method of claim 22, wherein the transfection reagent is lipid-based,polymer-based, or peptide-based.
 24. The method of claim 23, wherein thetransfection reagent comprises a charged polymer or a cell-penetratingpeptide.
 25. The method of claim 23, wherein the transfection reagentcomprises a cationic lipid, a liposome, or a micelle.
 26. The method ofclaim 6, wherein the medium is substantially free of immunosuppressants.27. The method of claim 4, wherein the first synthetic RNA molecule, thesecond synthetic RNA molecule, or both the first synthetic RNA moleculeand the second synthetic RNA molecule comprise at least onenon-canonical nucleotide selected from the group consisting of: a5-methyluridine residue, a pseudouridine residue, a5-methylpseudouridine residue, a 5-hydroxyuridine residue, a5-hydroxypseudouridine residue, and a 5-methylcytidine residue.
 28. Themethod of claim 4, wherein the first synthetic RNA molecule, the secondsynthetic RNA molecule, or both the first synthetic RNA molecule and thesecond synthetic RNA molecule further comprise one or more of a 5′-cap,a 5′-cap 1 structure, and a 3′-poly(A) tail.
 29. The method of claim 4,wherein the DNA repair template comprises one or more regions ofhomology to the DNA of the cell upstream or downstream of thesingle-strand break or the double-strand break.
 30. The method of claim29, wherein the DNA repair template comprises one or more regions ofhomology to the DNA of the cell upstream of the single-strand break orthe double-strand break.
 31. The method of claim 29, wherein the DNArepair template comprises one or more regions of homology to the DNA ofthe cell downstream of the single-strand break or the double-strandbreak.
 32. The method of claim 4, wherein the DNA repair templatecomprises one or more regions of homology to the DNA of the cellupstream and downstream of the single-strand break or the double-strandbreak.